ENZYMES AND ENZYME REACTIONS

All objectives listed are in the cognitive domain unless otherwise noted. The student, at
the end of the instructional period, is responsible for meeting these objectives by
achieving a cumulative score of 70% or better on all problem sets, case studies,
major exams, quizzes, and library assignments.

Objectives are indicated by a number followed by a purposeful question or statement.
The student, upon completion of the classroom component of clinical biochemistry,
will be responsible to successfully:

01     DEFINE AND/OR DISCUSS AN ENZYME

An enzyme is a complex protein that acts as an organic catalyst, is produced by living
cells, and is capable of acting independently.
(1)    it catalyzes chemical reactions in the cell.
(2)    causes an increase in the rate at which biological reactions occurs.
(3)    are not changed nor altered in the reaction.
(4)    do not change the equilibrium constant of a reaction.
(5)    does not change the thermodynamic properties of a reaction.
(6)    is a protein of variable molecular weights (MW). (Examples: ribonuclease has
         a MW of 13,700 and the MW of pyruvate dehydrogenase is 10,000,000.)

Enzymes catalyze chemical reactions that enable reactions to proceed at a rapid rate or to initiate reactions that would not take place otherwise.  They can be isolated, sometimes crystallized and denatured by heat.  They are sensitive to the presence of microorganisms and can be inactivated as they can be by heavy metals, detergents, and changes in pH.   There are well over 1,500 different enzymes that have been isolated and identified.  The presence of an enzyme in a body fluid such as plasma or serum is so minute that it cannot be measured by many conventional methods of protein assays, BUT the capacity of that enzyme to carry out an enzymatic reaction can be measured.  Enzyme activity can be measure in three different ways:
A.    Measuring the decrease in substrate.
B.    Determination of the amount of product formed.
C.    Follow the utilization of the substrate or the formation of the reaction
        product serially during the enzyme reaction. 

02    EXPLAIN THE NOMENCLATURE OF THE INTERNATIONAL UNION OF
BIOCHEMICAL ENZYME CLASSIFICATION SYSTEM

This organization has established a system of six major enzymes in which all enzymes
are categorized. Each major class can be subdivided into a major subclass with
subsequent sub-subclasses. Each enzyme will have a minimum of a four digit number,
plus it name. Look at NAD oxidoreductase as an example. It belongs to the class oxido-
reductase and is given the number designation of [1.]. The next subclass identifies the
substrate upon which the enzyme acts, in this case it is the alcohol groups of the substrate
which acts as an electron donor. It is given the number [1.]. The number designation is
now [1.1.]. Because the acceptor molecule is NAD (a coenzyme), it is given the number
of [1.]. The number designation becomes [1.1.1.]. A final number is included to identify
the specific enzyme and in this case is [1.]. The number classification for NAD:oxido_
reductase is 1.1.1.1. Other examples are:
( 1 )    Xanthine:oxygen reductase = 1.2.3.2.
( 2 )    D-glucose-6-phosphate phosphorylase = 3.1.3.9.

03    WRITE THE NAME OF THE FIRST CLASS OF ENZYMES, LIST ITS SEVEN
MAJOR SUBCLASSES, AND THE GENERAL REACTIONS THEY CATALYZE

Oxidoreductases:  (number designation "1."). Catalyze oxidation-reduction reactions.
(1)   dehydrogenases: removes electrons from donor hydrogens.
(2)   oxidases: transfers electrons of donor molecules to oxygens, and
        H₂O₂ is usually formed.
(3)   reductases: speeds addition of electrons to a donor molecule, reducing the
        positive valence.
(4)   perioxidases: uses peroxides as an oxidant (the molecule accept an electron),
       not oxygen.
(5)   catalases: detoxifies peroxides
(6)   oxygenases: incorporates oxygen into a substrate.
(7)   hydroxylases: oxidizes two hydrogen donor molecules and incorporates
        oxygen into one of the donor molecules.

04    WRITE THE NAME OF THE SECOND CLASS OF ENZYMES, LIST SEVEN
MAJOR MOIETIES THAT ARE TRANSFERRED AND FOUR MAJOR SUBCLASSES
ALONG WITH THE GENERAL REACTIONS THEY CATALYZE

Transferases: (number designation "2."). Transfers functional groups between donor
and acceptor molecules.  The major subclasses and the moieties that are transferred are:
( 1 )   aminotransferases: transfer of an amino group (-NH2) from an amino acid to
        a keto-acid receptor to form a new amino acid and keto acid.
( 2 )   kinases: these are phosphorylating enzymes, transferring the phosphoryl group
        from ATP to an alcohol or amino receptor.
( 3 )   glycosyl transferases: transferring a glucose group from one molecule to
        another.
( 4 )   acyl transferases: transfer of an acetic acid group from one molecule to another.
( 5 )   methyl transferases: transferring a methanol group from one molecule to
        another.
( 6 )   carboxyl transferases: transfer of a carboxyl group from one molecule to
        another.
( 7 )   carbonyl transferases: transfer of the carbonyl group in aldehydes, ketones,
        and carboxylic acids to another molecule.
( 8 )   phosphomutases: transfers phosphate groups

05    WRITE THE NAME OF THE THIRD CLASS OF ENZYMES, LIST NINE MAJOR
SUBCLASSES, AND THE GENERAL REACTIONS THEY CATALYZE

Hydrolases: (the number designation "3."). These are a special group of transferases
that cleaves certain bonds and adds water. The bonds cleaved are "C - O", peptide
bond "C - N", "O - P", and sulfide bond "C - S".
( 1 )   Esterases: catalyzes the hydrolysis of esters.
( 2 )   Glycosidases: catalyzes glycocytic linkages
( 3 )   Peptidases: converts peptides into amino acids.
( 4 )   Phosphatases: catalyze the hydrolysis of phosphoric acid esters.
( 5 )   Thiolases: affects thiolytic cleavage
( 6 )   Phospholipases: catalyzes the hydrolysis of a phospholipid.
( 7 )   Amidases: catalyzes the hydrolysis of amides.
( 8 )   Deaminases: catalyzes the removal of an amino group from organic
        compounds.
( 9 )   Ribonucleases: catalyzes the depolymerization of ribonucleic acid (RNA) with
       formation of mononucleotides.

06  WRITE THE NAME OF THE FOURTH CLASS OF ENZYMES, LIST SIX MAJOR
SUBCLASSES, AND THE GENERAL REACTIONS THEY CATALYZE

Lyases: (the number designation "4."). These enzymes add or remove elements of
water, ammonia, or carbon dioxide. They can also break "C - C", C - S", or certain
"C - N" bonds.
( 1 )    decarboxylases: removal of CO2 from α- and β-keto acids or amino acids.
( 2 )    Aldolases: catalyzes fructose-1,6-bisphosphate to D-glyceraldehyde-3-phosphate
         and dihydroxyacetone
( 3 )    Hydratases: catalyzes the breakage of C=O bonds, eliminating water.
( 4 )    Dehydratases: removal of water molecule in dehydration reaction.
( 5 )    Synthases: joins two molecules by cleaving a high energy phosphate bond
( 6 )    Lyases: removes groups from substrates by reactions other than hydrolysis or
        oxidation-reduction reactions.

07    WRITE THE THE NAME OF THE FIFTH CLASS OF ENZYMES, LIST FOUR
MAJOR SUBCLASSES, THE GENERAL REACTIONS THEY CATALYZE

Isomerases: (the number designation "5."). These catalyze the isomerization
 (conversion of one chemical substance into another) of its substrate.  Isomers
are molecules with the same molecular formula, but with different structure and
properties. Examples: [1] glucose and mannose, [2] pentane and isopentane.
(1)    Racemases: catalyzes the production of an optically active compound.
(2)    Epimerases: catalyzes the production of geometric isomers.
(3)    Isomerases: catalyzes intramolecular rearrangements.
(4)    Mutases: intermolecular transfer of groups such as phosphate.

08    WRITE THE NAME OF THE SIXTH CLASS OF ENZYMES, LIST TWO MAJOR
SUBCLASSES, THE GENERAL REACTIONS THEY CATALYZE

Ligases: (the number designation "6."). These forms bonds between two molecules at the expense of an ATP.
(1)   Synthetases: joins two molecules by splitting off a high-energy phosphate group.
(2)   Carboxylase: catalyzes the removal of the carboxyl group from an amino acid.

09   DEFINE THE TERM "REACTANT"

It is any substance that enters into a reactions and undergoes change. In enzyme
reactions, the reactant is the substrate.

10    EXPLAIN "KINETICS" AND WRITE ONE SYNONYM FOR IT

Synonym = energetics. Kinetics is the study of the rate of change of the initial state of
reactants and products to the final state of reactants and products. It is normal to drop
the use of the word "rate" and substitute "velocity". Velocity (or rate) describes the
change in the concentration of substrate or product in a given time. Velocity is a term
that measures how fast the substrate disappears and the products appear in a unit of time.

11    EXPLAIN THE CONCEPT OF ORDER IN A CHEMICAL REACTION

The word "order" describes how a reaction goes. It is a mathematical expression.
There are four type of "order" reactions.
( 1 )   First order reaction. This is a reaction rate that is exactly proportional to the
concentration of one reactant. If you add more reactant, the velocity of the reaction
speeds up.  If less reactant is use, the velocity of the reaction slows. It means that all
of the enzyme is NOT tied up in the Enzyme——>Substrate (E—> S) complex.
( 2 )   Second order reaction. The rate of this reaction is proportional to the product
of the concentration of the two reactants. If you add more reactants, the rate of the
reaction is increased.
( 3 )   Third order reactions. These are rare reactions. The rate of this reaction is
proportional to the product of the three concentration terms.
( 4 )   Zero order reaction. This is a reaction rate that is independent of its reactants
(substrate).  This reaction occurs when all of the enzyme in the reaction is tied up in
the E ——> S complex.

12    DESCRIBE HOW ENZYMES ARE MEASURED

A.    Enzymes may be measured in terms of their activity.  The enzyme is measured in terms of their activity by measuring the amount of appropriate substrate that can be converted into product in a specific unit of time under strictly defined and controlled conditions.  The procedure may be set up with a large concentration of substrate and because of the test conditions, a small portion of substrated is consumed and measured.

B.     Enzymes may be measured in by determining the amount of metabolite that is consumed in the reaction.   In this case the metabolite is the substrate.   The reaction may allowed to continue until all the metabolite is consumed. 

13    EXPLAIN THE CONCEPT OF A STANDARD UNIT OF ENZYME ACTIVITY

Enzyme activity is usually expressed in units of micromoles (μmoles) of substrate
converted into product per minute under specified conditions. A standard unit of
enzyme activity is that amount of activity that catalyzes 1.0 μmole of substrate
per minute.  The defined conditions include temperature, pH, the buffer used,
substrate concentration, and cofactor concentration.

In clinical chemistry, the enzyme activity in body fluids (serum and plasma) is
related to a volume of 1.0 mL, therefore the results may be reported in milliunits
(mU) per mL, where a mU is equal to mμmoles of substrate transformed per
mL × minutes.   In some laboratories and procedures, the reference volume is a
liter instead of a mL. 

14    EXPLAIN THE CONCEPT OF THE INITIAL VELOCITY OF AN ENZYME-
CATALYZED REACTION

The initial velocity of an enzyme-catalyzed reaction is dependent upon the
oncentration of substrate. If the [substrate] is increased, the rate of the reaction
(its velocity) will also increase. The velocity can increase until all the enzyme is
saturated by the substrate. The rate (velocity) at which an enzyme reaction "goes" is
dependent upon the concentration of enzyme and substrate. See following illustration.
  

15    EXPLAIN THE EFFECT OF TEMPERATURE UPON ENZYME RATES

Optimum temperature for most enzyme reactions in mammalian systems lies
between 40 ^OC and 45 ^OC. Temperature effects follow a bell-shaped (Gaussian)
curve.   The effects of temperature on enzyme activity are complex and are
interrelated with the pH, buffer system, and substrate concentration.
   
In general the following observations are applicable.  Denaturation begins around
 50 ^OC.   A temperature of 60 ^OC generally results in the inactivation of most all
enzymes.  Between 0 ^OC and 40 ^OC, enzyme activity is slow, but with every
10 ^OC increase in temperature, enzyme activity will increase by a factor from
1.5 to 2 times.  This means that a temperature increase of 1.0 ^OC will change
the activity of the enzyme from 4 to 10%.  In cases of  hypothermia, enzyme
activity is decreased and there is a corresponding decrease in oxygen demand.
Hypothermia in humans is present between 32 ^OC and 35 ^OC.

16    EXPLAIN THE EFFECT OF pH UPON ENZYME STATES AND LIST A MINIMUM
OF FOUR EXAMPLES

Optimum pH varies for all protein molecules, including enzymes. Most enzymes
are active between a pH of 4.0 and 8.0. The different side groups determines the
degree of ionization, which determines the degree of enzymatic activity. The
ionized state of the substrate determines the ability of enzymes to bind to it.
Some enzymes are active over a wide pH range and others over a narrow range.
The nature of the buffer and the substrate concentration also affects pH effects
upon the enzyme.  The effects of pH follow a bell-shaped (gaussian) curve as
follows:
       

Examples of the optimum range of selected enzymes: [1] pepsin (pH = 1.5),
[2] arginase (pH = 9.7), [3] acid phosphatase (pH = 6.2), [4] creatinine
kinase (pH = 8.0), [5] trypsin (pH = 8.0), and [ 6 ] cholinesterase (pH = 7.7)
[7]  pancreatic lipase (pH = 8.0],  [8]  urease (pH = 7.0),  [9]  Maltase (pH =
6.1 -- 6.8),  [10]  Catalase  (pH =  7.0)

17     DESCRIBE HOW SUBSTRATE CONCENTRATION AFFECTS ENZYME ACTIVITY

Within limits, the rate (velocity) of an enzyme reaction will increase as the
concentration of the substrate increases.  It is a fact that when the substrate
attains a certain concentration the velocity of the reaction will not increase
and the reaction has reached a maximum.  This is dependent that the enzyme
concentration remains constant.  Visualize (see illustration below) a single
enzyme surrounded by eight substrate molecules.   This is a low substrate-
enzyme concentration and the number of substrate molecules reacting with the
reaction site of the enzyme will be decreased yielding a low rate of formation of
the enzyme-substrate complex.  If the single enzyme is surrounded by many
substrate molecules, the substrate-enzyme concentration is high and this results
in an increased rate of formation of the enzyme-substrate complex. This is
because the time to fill the emptied binding site of the enzyme becomes shorter.
An excess of substrate will result in a fixed (maximum) rate of activity because
the reaction sites of the enzyme(s) are saturated and cannot be filled any
faster.  It is this concept that has resulted in developing the mathematical
concept of the enzyme reaction. 

In developing enzyme concepts, high substrate concentrations are not utilized
because it has been shown that such concentrations can inhibit  the reaction.

18    DISCUSS THE ROLE OF BUFFERS IN ENZYME ASSAYS

Laboratory assay procedures utilize some type of a buffer. As the enzyme reaction proceeds, the product formed may affect the pH in the test solution which may affect the rate of the reaction. Buffers are selected for test procedures that will hold the pH constant (with minimum change). Ideally the buffer used should always be at its lowest concentration because some buffers at a high concentration may compete with the substrate for the enzyme, producing an inhibitory effect on the reaction rate. This has been demonstrated using NADP dependent lactic acid dehydrogenase. If TRIS buffer is used in high concentrations, it forms an enzyme • buffer complex, removing theenzyme from the reaction system and slowing the velocity of the reaction down.

There are test situations in which the buffer is a part of the reaction process and facilitates the reaction as with alkaline phosphatase assays. In this alkaline phosphate assay reaction, p-nitrophenol phosphate is used as a substrate (this is a buffer) and a second buffer: AMP buffer (2-amino-2-methyl-1-propanol • AMP). This buffer system holds the pH at 10.2. As alkaline phosphatase hydrolyzes the substrate to n-phenol and phosphate, the AMP-buffer acts as a phosphate acceptor, removing it from the system. This dual buffer and acceptor system enhances the reaction rate and produces a better enzyme reaction. If glyclglycine buffer would be used, it will maintain the pH at 10.2, but contributes nothing else to the reaction. The reaction using glycylglycine buffer is inhibited when contrasted with the AMP buffer system which has an augmenting effect.

19    DEFINE COFACTORS AND LIST SEVERAL EXAMPLES

Cofactors, for the purpose of this course are considered to be organic molecules needed by the enzyme for enzymatic activity.  These molecules are also called coenzymes  which unite with another molecule (enzyme) in order for a reaction to take place.  Generally coenzymes have a low molecular weight, but they have very specific functions.   For example in the illustration below, NAD and NADP in dehydrogenase reactions will transfer hydrogen ions.  There are two types of coenzymes: cosubstrates and prosthetic groups.  A cosubstrate will bind with the enzyme but is altered in the reaction, dissociates from the reaction site, and is regenerated.  A prosthetic group, also binds with the enzyme, but must return to its original form.  Thiamine (vitamin B₁) is a cofactor.  It is first converted to the coenzyme thiamine pyrophosphate (TPP), where it can be used in a decarboxylation reaction with the enzyme α-ketogluterate dehydrogenase. Look at the following example using this cofactor.
                   
Other examples of cofactors or coenzymes  are:
A.    Niacin, a vitamin source for coenzymes nicotinamide adenine
        dinucleotide [NAD⁺] and nicotinamide adenine dinucleotide phosphate
        [NADP⁺],  are involved in oxidation reduction reactions involving two-
        electron transfers.  It role is that of a cosubstrate.
B.    Riboflavin (B₂), a vitamin source for coenzymes flavin mononucleotide
        [FMN] and flavin adenine dinucleotide [FAD], are involved in oxidation
         reduction reactions involving one- and two-electron transfers.  Its role is
         that of a prosthetic group.
C.   Pyridoxine ((B₆), a vitamin source for coenzyme pyridoxal phosphate [TPP],
        is invovled in transfer fo two-carbon fragments that contain a carbonyl group.
        It role is that of a prosthetic group.
D.    Vitamin A, the vitamin source for retinal, has a major metabolic role in
         vision.  Its role is that of a prosthetic group.
E.     Pantothenate (B₃), the vitamin source for coenzyme A (CoA), is involved in
         the transfer of acyl groups.   Its role is that of a cosubstrate.

20    DEFINE ACTIVATORS, AND LIST A MINIMUM OF FOUR EXAMPLES

Activators are generally deemed to be metal ions and have bee referred to as inorganic complements.. This term may be considered to be synonymous with cofactors.
A.    Magnesium, an essential dietary mineral and divalent cation, is required for
        kinases and form a complex with adenosine phosphates to act a a shield for
        the negatively charged phosphate groups to , is involved as an activator  in
        the ATP phosphate transfer reactions. 
B.    Zinc, a divalent cation, acts as a cofactor that binds to the side-chains of three
        histidine residues.  Zinc binds in a molecule of water which  ionizes permitting
        activation of a carboxyl group into a nucleophilic hydroxide iron that attacks
        the substrate.
C.    Iron, a divalent cation, is part of the heme structure in the enzyme catalase,
        which catalyzes H₂O₂.
D.   Amylase is activated by chloride ion and pyruvate kinase requires both
        magnesium and potassium. Examples of other activators are (1) copper,
        (2) cobalt, (3) manganese, and (4) calcium.
E.    Manganese (Mn⁺⁺⁾ is essential for peptidase reactions and for phosphate
        transferring enzymes.

Inhibitors can be removed by chelation.   Ca⁺⁺ and Mg^++, which can inhibit hexokinase, can be removed by either EDTA or oxalate, in which case the inhibition effect is removed.  Another inhibitor mode of action is to interact with the enzyme by binding up the reaction site of the enzyme and prevent substrate binding.  Some inhibitors can act by attaching to the enzyme at a different site to form a complex and alter the configuration of the enzyme (allosteric effect) and prevent enzyme activity.  Inhibitors are classified into three different groups:
A.    Competitive inhibitors that bind with the active site.  See Objective 24.
B.    Noncompetitive inhibitors that bind at a allosteric site.  See Objective 25.
C.    Uncompetitive inhibitors that bind with the enzyme-substrate complex.  See Objective 26.

It is to be noted that when doing enzyme experiments and tests that an excess of these inorganic activators may have an inhibitory effect upon the reaction.

21   DISCUSS WHAT IS HAPPENING ENERGY-WISE WHEN SUBSTRATE IS BEING CONVERTED TO A PRODUCT

The following equation expresses what happens to substrate and enzyme in a
 typical reaction:
           
The E • S complex is a transition state and can reverse itself.  When the reaction goes
forward and is complete, the product is formed.  The enzyme is not used up in the
reaction and will return to original form when the reaction is concluded.

Examine the following energy diagram that simply reflects the effects of a catalyst.
   
(1)   This magnitude of energy must be added to the system to convert substrate to
        product. This is called energy of activation, which overcomes the barrier that
        tends to block the reaction from being initiated.
(2)   The enzyme lowers the amount of energy needed for substrate to form the
        product. It represents the ease by which E + S  ―>  E • S.
(3)    This is the amount of free energy that passes into the system because of the
        conversion of the E • S complex being converted into the product.
(4)   This represents the energy released by the non-enzyme reaction. This much
        energy must be put back into the product to reverse the reaction.
(5) This is the amount of energy released by the enzyme reaction

22    EXPLAIN THE PURPOSE OF ENZYME KINETIC FORMULAS

In the early beginnings of enzyme studies, it was recognized that in regular chemical reactions, kinetic principles could be generally applied to enzyme kinetics. They noticed a feature appearing in that the amount of substrate added to the reaction affected the rate. It was also noted that at an increased substrate concentration, the initial reaction rate falls away. This resulted in the development of rate (velocity) equations to describe the velocity of the reaction as it related to the concentration. In 1913, the first equation was developed to describe enzyme kinetics by L. Michaelis and M. L. Menton.

23    DISCUSS AND/OR DERIVE THE MICHAELIS AND MENTEN EQUASTION

This equation assumes that the following reaction takes place:
      
01    The following are also assumed:
        A.    Reactions are reversible.
        B.     During the initial part of the reaction, the E • S complex formation is
                constant.
        C.    All of the enzyme is involved in the reaction, none of it is free.
        D.    The rate of the reaction is maximal.
        E.    There is a one-substrate and one-enzyme reaction.

This equation relates the initial reaction velocity of E —> P to the (a) maximum reaction velocity, (b) the substrate concentration, and (c) the dissociation constant for the E • S complex.

The Michaelis-Menten Equation is
                    V_max
         V = ---------------
                  K_m + [S]

A.     Vmax is "velocity of reaction" and expression of enzyme activity.
         a.    Its value is [S] and [E] dependent. This value is plotted on the ordinate.
B.      V is the initial velocity of the reaction.
C.     The maximum rate of enzyme activity is reached when the available enzyme
          is saturated with substrate.
D.     K_m is the Michaelis-Menten constant. It is a mathematical expression of the rates of formation of E ⋅ S complexes and product.
                    k₂ + k₃
        K_m = --------------       This equation come from (1) above.
                        k₁

Note that k₄ is very small, an insignificant value and is dropped.

E. K_m can be manipulated down to represent [S]
F. Additional mathematical manipulations are possible that bring K_m to be equal to [S]. In this event, K_m will become [S] and this means that enzyme activity is occurring at ½ V_max. When the reaction is going at a rate that is independent of the [S] (but dependent upon the [E], this is termed as a zero order reaction. K_m is plotted on the abscissa.

Look at a typical Michaelis-Menten curve:
               
1.    This equation is difficult to use because it is based upon ideal enzyme behaviors. The fact is that enzymes do not follow expected behaviors. In some enzyme reactions, a high [S] inhibits the formation of E• S complexes.
2.     In this example, assume that V_max = 25 enzyme units
3.     Then ½ V_max = 12.5
4.     K_m = 2.0 x 10^-4 mol/L can be plotted on the graph by dropping a perpendicular line, after locating the point (at which ½ Vmax occurs on the ordinate) from the curve that was plotted to obtain V_max.

What has happened to the Michaelis-Menten equation, because of its difficulty to use, is that it has been converted to other reciprocal forms that are easier to use. Lineweaver and Burke mathematically manipulated the Michaelis-Menten equation to a reciprocal form that is easier to use.

24    DISCUSS THE LINEWEAVER-BURKE EQUATION/PLOT

These two men (Lineweaver and Burke) set up the Michaelis-Menten equation as a reciprocal equation (through a series of mathematical manipulations) as follows:
        1                K_m         1                1
        ----   =   ---------- • ---------  +  ---------
        V₀          V_max         [S]           V_max

The normal Lineweaver-Burke (L-B) plot is as follows:
       
            The values on the ordinate represents dimensions of moles/liter.  If the
            values move to the left the substrate is decreasing or if to the right, the
            substrate appears to be increasing.

A.    This plot is consistent with an uninhibited reaction.
B.    If the same procedure is repeated exactly, the same slope will be plotted.
C.    Using the data in Objective 21, if V_max = 25, then 1/V_max = 0.04. This value is
         to be plotted on the ordinate of the L-B plot.
D.    Using the data in Objective 21, if K_m = 2.0 X 10^-4, then 1/Km = -0.5 X 10^-4
E.    The slope for K_m / V_max can be calculated and plotted, it is easier to obtain
         K_m from the intercept on the abscissa.
F.    Draw a straight line from the points on the abscissa and ordinate.  The slope
        obtain is the same as if calculated from K_m / V_max.
G.    The L-B plot is most useful in evaluating enzyme inhibited reactions which will be reviewed in the next few objectives.

25    BRIEFLY EXPLAIN THE CONCEPT OF ENZYME INHIBITION

Enzyme inhibition occurs when the immediate product formed or the products formed by other enzymes slow down or stop the function of the enzyme. Medications can be prescribed that target an enzyme or enzymes to stop its action. An inhibitor can be an enzyme product or a medicinal drug. Inhibitors can act by one of the following mechanisms:
a.    mimic the substrate molecule, bind to the reactive site and block attachment to
        the substrate molecule.
b.    bind to a different part of the enzyme molecule and induce an allosteric change
       in the enzyme structure that disables the enzyme from attaching to its substrate.
c.    bind to a coenzyme or metal ion activator (Ca++, Mg++, Zn++, Mn++, etc)
       and block it from functioning. The enzyme may be able to form the
       enzyme • substrate complex, but without the coenzyme/activator, the
       reaction is stopped.
There are three major type of enzyme inhibition:
    (1)  competitive
    (2)  non-competitive
    (3)  uncompetitive



26    DISCUSS COMPETITIVE ENZYME INHIBITION AND DESCRIBE ITS CHARACTERISTIC L-B PLOT

In competitive inhibition, the competitor molecule binds at the same site as the substrate. Most enzyme inhibition reactions are of this type. The following illustration is an example of a normal reaction without inhibition. In this reaction the substrate is being used up so that there is a 1/[S] change and this will affect the plots on the abscissa (the x-axis).
       

Consider the following illustration for competitive inhibition.
           
a.    There is a build up of enzyme • inhibitor (E • I) complexes and the reaction
        is stopped.
b.    k₁ can be measured as velocity of E • I complex formation and plots can be
        made on the ordinate coordinates.
c.    The substrate concentration [S] (in a manner of speaking) increases because of
        the decreasing enzyme concentration.
d.    the values plotted on the abscissa coordinate will reflect this apparent increase
        in [S].
e.    If several inhibitor concentrations [I] are plotted, the same ordinate coordinate
       is obtained but different abscissa ordinates are generated.
f.    Examine the following L-B plot to compare competitive inhibition plots (based
       on changes in [I] with a normal reaction plot.
       

27    DISCUSS NON-COMPETITIVE ENZYME INHIBIITON AND DESCRIBE ITS CHARACTERISTIC L-B PLOT

In this type of inhibition, the inhibitor binds at a different site from that of the substrate. In this example, the substrate can bind to the enzyme to form the E • S complex, then the inhibitor bind to the complex. It is possible that the inhibitor can bind first to the enzyme and the substrate can attach to the E • I complex or it may not be able to form an attachment. In any of these possibilities, the reaction is stopped and no product is formed. The following illustration is an example of non-competitive inhibition where the substrate can bind to the E • I complex.
           
Changing the substrate concentration does not modify inhibitor binding or effects. There will be no changes in the abscissa coordinates, but there will changes in the ordinate coordinates. In this model, the substrate concentration decreases, causing the plots to fall above the plot for a normal slope without inhibition.  It is possible that some of the substrate is being converted to product and if this is so, then it will be at a slower rate and in low quantities.
  

28   DISCUSS UNCOMPETITIVE ENZYME INHIBITION AND DESCRIBE ITS CHARACTERISTIC L-B PLOT

This is a rare occurrence in single substrate reactions. It is common in two substrate reactions. Uncompetitive inhibition is characterized by the inhibitor binding to the substrate. It does not bind to the enzyme first. Examine the example of binding of inhibitor with substrate.
           
The typical uncompetitive L-B plot is the result of the binding of the inhibitor that initiates an effect in the substrate that produces a new slope (K_m/V_max) that will plot parallel to that of a normal slope. The substrate appears to be decreasing and it will give a small V₀ value, but K_m and V_max produce equivalent values. Examine the following L-B plot for uncompetitive inhibition.
 
       
29   DESCRIBE HOW AN ACTUAL ENZYME CAN BE INHIBITED USING TRYPSIN AS A MODEL

Trypsin is a globular protein that belongs to a group of proteolytic enzymes called the serine proteases. (The serine molecule has a role in the reaction.)   Serine proteases make up several of the clotting factors (II, VII, IX, X, XI, XII, XIII).  The trypsin molecule has a active site that accepts an arginine side-chain. Trypsin cleaves peptide bonds that has the adjacent basic amino acid arginine. Benzamide is a competitive inhibitor for trypsin. To the trypsin molecule, benzamide is a "look-a-like" to arginine. See following illustration:
       
Trypsin can form a reversible bond with benzamide. In this type of inhibition, it is possible to reverse this type of inhibition because covalent bonds are not formed. It is possible for the three types of inhibition (competitive, non-competitive, and uncompetitive) that the inhibition effects can be reversed. If the inhibitor can form a covalent bond(s), then inhibition becomes non-reversible. See following example of reversible inhibition for trypsin.
           
Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of trypsin. DFP also inhibits chymotrypsin and elastase. An enzyme that has an available and exposed serine molecule will form a covalent bond with DFP. For trypsin, serine (with its exposed alcohol group) is located on position #195 of the protein chain. DFP is highly toxic and used to treat glaucoma. It can be labeled with 32P to measure RBC mass and life span. It also inhibits acetylcholine by converting it to acetic acid and choline, which turns off synaptic activity in nerve tissue and motor end-plates. DFP can be used to make deadly war gases. See following for how DFP interacts with serine.
       

30    EXPLAIN IN GENERAL TERMS HOW DRUGS MAY ACT AS INHIBITORS

The concept of drug therapy is based upon enzyme inhibition. Drug research is designed to (1) inhibit a specific enzyme in a specific metabolic pathway and (2) exhibit high toxicity to pathogens and tumor cells, but a low toxicity to healthy cells. Drugs that can kill unhealthy cells can also kill healthy cells. One of the strategies in drug research that investigators try to take advantage of is the rapid generation time of undesirable cells (pathogens and tumor cells). This rapid rate of reproduction tends to make these cells more sensitive to drug therapy than healthy cells.

31    DESCRIBE HOW THE DRUGS SULFONAMIDE, METHOTREXATE, AND ISONIAZID ARE USED IN MEDICINE AS INHIBITORS

These drugs are representative of how medical treatment is effected through enzyme inhibition.
Sulfonamide were the first drugs used in modern chemotherapy. Sulfanilamide is the simplest of this class of drugs. It is an analog of p-aminobenzoic acid (PABA). Compare their structures:
       
PABA is a precursor in the synthesis of folic acid. Bacteria must first synthesize folic acid in order to produce dihydrofolate. Bacteria are not capable of incorporating folic acid from the host. Since sulfanilamide is an analog of PABA, bacteria accept this substitute and make dihydropteroate. Bacteria cannot grow or divide without folic acid... they then die. Sulfanilamide levels in the human body as an antibiotic are toxic to the microorganism, but is well tolerated by human cells.

Methotrexate is the structural analog of folate and has the property of binding 1000 times stronger to the enzyme dihydrofolate reductase than does folate. Folate is a coenzyme required for the transfer of one carbon units in the synthesis of purines and pyrimidines. In the presence of methotrexate, the synthesis of thymidine monophosphate ceases and this causes a termination of thymidine (needed for cell division). It has been found to be an effective treatment for childhood leukemia. One problem with this inhibitor.... the genes that produce dihydrofolate reductase, in the presence of methotrexate, can amplify their production of the enzyme and cancel out its toxic effects. See following illustration for folate and its analog.
           
Isoniazid is the structural analog of nicotinamide. Nicotinamide is required for the synthesis of nicotinamide coenzymes and has been found to be effective in the treatment of tuberculosis (caused by Mycobacterium tuberculosis). This drug is thought to interfere with the lipid and nucleic acid metabolism of growing bacteria by causing defective development of the bacterial cell wall. See following illustration of nicotinamide and its analog.
           

                                                            NOTE
Pathogens and tumor cells can become resistant to drugs. One way of foiling the effects of the drug is for the cell to create a new pathway to bypass the inhibited pathway. A second strategy employed is to modify the pathway so it will produce larger quantities of enzyme to compensate for the inactivated enzyme.

32    BRIEFLY EXPLAIN WHY CELLS EXHIBIT ENZYME REGULATION

The cell is a complex system that carries out a large number of reactions. There must be some mechanism to control the intercellular concentration of its products to prevent detrimental effects from over- or under-production. The cell can regulate enzymes through (1) activation, (2) product inhibition, (3) allosteric effects, and (4) covalent activation.

33    EXPLAIN HOW GLUTATHIONE CAN FUNCTION TO ACTIVATE OTHER ENZYMES

Glutathione is a tripeptide (glutamine + cysteine + glycine) that is not a specific coenzyme, but does function as one for maleyl acetoacetate isomerase, prostaglandin PGE2 synthetase, and glyoxalase. It has the ability to reduce methemoglobin to hemoglobin. It also detoxifies by binding peroxides and chelates metals. It also serves as the substrate for other enzymes. Glutathione can activate and deactivate enzymes by oxidizing (causes to give up an electron) the sulfhydryl group of cysteine. This occurs when there is a disulfide interchange with disulfide bonds of other proteins to produce a stable functioning molecule. If glutathione is present, reactions are activated, if it is absent or in decreased concentrations, reactions do not occur. Other methods include (1) acting as a re-locating molecule to transfer a sulfur group and (2) it is used by other enzymes to transport amino acids across the cell membrane.

34    BRIEFLY EXPLAIN THE PRINCIPLE OF ALLOSTERIC CONTROL OF ENZYME ACTIVITY

Allosterism, also called allosteric control, is an elaborate form of enzyme regulation. In its simplest form, the allosteric enzyme has two reactive sites: one site for binding the substrate and the second site for the binding of the effector (modulator or modifier) molecule. Features of allosteric control include:
a.   allosteric enzymes contain more than one peptide chain.
b.   contain a catalytic center (where the reaction takes place) and at least one
       regulatory or effector center (where the regulatory molecule binds).
c.   can be subjected to positive control (the catalytic site can accept its substrate
      molecules allosterically) or negative control (the catalytic site is closed off
      allosterically to its substrate molecules).
d.   coenzymes, substrates, or products can act as effectors.
e.   can undergo change in conformation when influenced by the proper effector
      (coenzyme, substrate, or product).

35    DISCUSS "FEEDBACK CONTROL" USING THE INFORMATION OF ISOLEUCINE FROM THREONINE AS AN EXAMPLE

This is an unusual type of inhibition. Kinetic studies are atypical and does not categorize this type of inhibition as competitive, non-competitive, or uncompetitive. Isoleucine is a specific inhibitor, something not usually observed with amino acids or related compounds. Other names for this type of inhibition are (1) end-product inhibition, (2) feedback inhibition, and (3) retro-inhibition. Note the reaction sequence....
       
    E1  =  threonine dehydratase
    E2 and E3  =  intermediate step enzymes
    E4  =  dehydratase
    E5  =  transaminase

36    DISCUSS "PRODUCT INHIBITION" USING THE INFORMATION OF GLUCOSE-6-PHOSPHATE FROM GLUCOSE AS A MODEL

This is thought to be a type of competitive inhibition. This is a single reaction step as follows:
       
As the glucose-6-phosphate concentration increases, it exerts a negative effect upon the reaction by slowing down the rate of enzyme ⋅ substrate complex formation. It is thought to "hinder" the catalytic site. Physiologically, inside the cell where this reaction occurs, it is exposed to Pi and AMP, both of which have a counteracting effect upon the inhibitory effect of glucose-6-P. The enzyme that drives this reaction (hexokinase) has a high affinity for glucose, which tends to resist the competitive inhibitory effect of glucose-6-P.

37    DISCUSS ALLOSTERIC PHENOMENON USING HEMOGLOBIN AS A MODEL OF A REGULATORY PROTEIN

Hemoglobin (Hgb) is NOT an enzyme. It is a protein and it can act as an enzyme. Its major function is to transport oxygen to the tissues. The principles by which hemoglobin binds oxygen is considered to be similar to the binding of substrate to an allosteric enzyme. The hemoglobin unit is composed of:
        a.     two α₁ subunits of 141 amino acids each.
        b.     two β₁ subunits of 146 amino acids each.
Each subunit can bind one molecule of oxygen. In the lungs, hemoglobin is saturated with oxygen. The lung's environment is characterized by a high oxygen tension that favors the binding of Hgb. As Hgb binds to one of the subunits, that subunit stimulates the binding of oxygen to the second subunit, etc. until all the subunits are bound with oxygen. This is allosterism. In the tissues, hemoglobin (under normal conditions) will release ½ of its oxygen. Examine the following illustration for the 3-dimensional appearance of the α₁ and β₁ subunit. The "star" in the illustration represents the binding site of the oxygen molecule.
               
X-ray diffraction studies of hemoglobin provides the following facts:
a.    The Hgb molecules exists in two different states (oxyhemoglobin and
         (deoxyhemoglobin).
b.    Hgb is a dimer, that is, it has two different halves.
c.    The Hgb molecule consists of two dimers.
d.    Each dimer consists of an α₁ and β₁ subunit that is tightly bound to each other.
e.    The dimers are capable of motion with each other.
f.    The moveable dimers are held together by salt bridges. When the Hgb molecule
        is in its "deoxy" configuration, hydrogen bonds hold it together.

                                                                NOTE
Salt bridges are ionic bonds. These bridges forms between amino acid side chains that contain positive and negative charges. Histidine side chains have a major role in helping to stabilize the Hgb molecule in its "deoxy" configuration.

Additional chemistry studies of hemoglobin disclose these occurrences:
a.     In the tissues, after giving up its oxygen molecules, glycerate-2,3-bisphosphate
        (GBP) binds to Hgb and lowers it affinity for oxygen. GBP will bind in the
        spaces between the subunits of the Hgb dimer. See following illustration
        for GBP.
           
b.     Carbon dioxide (in the presence of carbonic anhydride) can combine with
        water to form carbonic acid (unstable) and dissociates to hydrogen and
        bicarbonate ion. The bicarbonate ion will bind with sodium, forming sodium
        bicarbonate (a principle blood buffer) that will bind to Hgb, increasing it s
        tability in the "deoxy" form.
c.     When "deoxy" Hgb is returned to the environment of the lungs, it is converted
         to the "oxy" form of Hgb.
d.     Oxygen binds to heme with it ferrous iron atom (Fe⁺⁺). Fe⁺⁺ has a high
        affinity for oxygen. The ferric state (Fe³⁺) has the wrong electronic structure
         to bind with oxygen. Fe⁺⁺ can shift within the porphyrin ring to assume a
        more favorable position for bonding.
e.     The Fe⁺⁺ ion makes four single bonds to the nitrogen's in the heme ring. There
         is a fifth bond to the histidine group in the Hgb molecule. The sixth and last
         bond site is for oxygen.
f.     When oxygen bind to the Fe⁺⁺ ion, the ion shifts and induces a strain and
        subsequent movement in the subunit that affects the dimer. Stress in one
        subunit stresses the other subunit, making it more receptive of the oxygen
        molecule. When the first dimer is filled, the stress is transferred to the second
        dimer facilitating addition of the oxygen molecule. This process continues
        until all four subunits are filled with oxygen. When this occurs, the Hgb is in
        its "oxy” hgb form. Refer to the following illustration for the sequential model
         for hemoglobin allosterism.
       
Look in more detail at what happens in the hemoglobin molecule when conditions are imposed.
a.     If the hydrogen ion increases, lowering the pH, the affinity of Hgb for oxygen
         decreases.
b.     If the pH = 7.6 (as might occur in the lungs), 02 tension = 40 mm Hg, Hgb
        can saturate with 02 to 80% of its capacity.
c.     If the pH holds at 7.6 and 02 tension increases to 80 mm Hg, then Hgb will
        saturate from 95% to 98% of its capacity.
d.     If the pH drops to 6.8 (as might occur in the tissues), then Hgb can retain
        only about 45% of its oxygen.
e.     In the tissues, the 02 tension decreases and this results in Hgb giving up much
        of its oxygen. Metabolically active tissues have increased CO2, low 02 tension,
        increased [H+], and increased glycerate-2,3-bisphosphate (GBP). GBP is also
        known as 2,3-diphosphoglycerate (DPG). These three factors decrease the
        affinity of Hgb for oxygen. GBP is found in higher concentrations in the
        RBC's of animals living at higher altitudes.
f.     The influence of C02 and H+ on the release of 02 is called the Bohr effect.

                                                                        NOTE
CO₂, H⁺, O₂, and GBP may be called ligands.  A ligand is any molecule that binds to a macromolecule.   Ligands can be ions, inorganic or organic molecules, or small, low molecular weight proteins (as glutathione).

In the lungs, the following occurs that affects the allosterism of Hgb.
a.   pO₂ is increased and pCO₂ is decreased.
b.   The binding of oxygen to hemoglobin produces an increase in H⁺, which tends
       to decrease the pH. The released hydrogen ion is captured by the bicarbonate
       buffer system and removed preventing the pH drop.
c.   The binding affinity of GBP to Hgb decreases, resulting in its displacement from
       the central cavity of the Hgb dimer (located between the β₁ subunits). It can
       become substrate in the glycolytic pathway.
d.   The loss of CO₂ and H⁺ from Hgb decreases the acidity of Hgb.
e.   Because of the association of the lungs with the external environment, there is
      a small decrease in temperature, which favors the association of oxygen to the
      heme groups.
f.   The shift in pH in the lungs with increased formation of oxyhemoglobin is part
      of the Bohr effect and is expressed by the following equation:

H •Hgb  +  O₂  ——>  Hgb • O₂  +  H⁺

In the tissues, the following occurs that produces effects in the opposite direction.
a..     pO₂ is decreased and pCO₂ is increased.
b.     Lactic acid is increased (lowering the pH), helping to facilitate the release
        oxygen from Hgb.
c.     The loss of oxygen from Hgb allows Hgb to bind CO₂ and H⁺. This increases
         the acidity of Hgb, part of the Bohr effect.
d.     The increased levels of GBP in the tissues favors binding into the Hgb dimers
        and promotes the release of oxygen.
e.     There is a corresponding increase in temperature, also favoring release of
        oxygen and increasing the formation of deoxyhemoglobin.

The following oxygen dissociation curve for hemoglobin the shifting as oxygen is absorbed and discharged from the Hgb molecule. Note that (1) represents a shift to the left favoring formation of oxyhemoglobin and (2) represents a shift to the right, favoring the formation of deoxyhemoglobin.
           

38    DESCRIBE ALLOSTERISM AFFECTING ENZYME CONFIGURATION USING ASPARTATE CARBAMOYLTRANSFERASE AS AN EXAMPLE

This enzyme is one of the best studied allosteric enzymes of Escherichia coli. This reactions which is the first step in the synthesis of cytidine triphosphate is a follows:

carbamoyl phosphate + aspartate --------------> carbamoyl L-aspartate

Schematically, the reaction goes like something this:
   

39      DIFFERENTIATE BETWEEN ENZYMES THAT ARE FOUND IN CELLULAR AND EXTRACELLULAR COMPARTMENTS AND THEIR SIGNIFICANCE.

Enzymes  that are found in plasma may be designated as plasma specific or non-plasma specific.   The plasma specific enzymes (such as ferroxidase, pseudocholinesterase or lipoprotein lipase) are synthesized in the liver and released into the plasma.  These three enzyme are useful in estimating liver function.  The coagulation enzymes also 'fit' into this category.  They are designated as 'plasma specific' because they are present in higher concentrations and and their activity occurs here.  The non-plasma specific enzymes fall into two categories and have little (if any) function  in the plasma.  One category is 'enzymes of secretion'  and are synthesized in the exocrine glands (such as the prostate, certain glands of the gastric mucosa, and pancreas).  Examples are amylase, lipase, acid phosphatase, and alkaline phosphatase.  If their concentration is decreased in the plasma, then there may be tissue damage or necrosis.  If the concentration is elevated, then there may be pathological significance.   The other category of "non-plasma specific' enzymes are designated as enzymes of metabolism.  Their concentration in the cellular environment is higher than in the plasma and if they are found in the plasma in significant amounts, then cellular damage is suspected.  Examples of these enzymes are creatine kinase, alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase.

40      DISUSS THE DISTRIBUTION OF ENZYMES IN THE CELL

The distribution of enzymes in the cell are not even throughout the cell.   The enzymes such as aldolase, alanine transaminase (ALT), and lactate dehydrogenase are found in the cytosol.   Glutamic dehydrogenase is found in the mitochondria, but not the cytosol.   Both malic dehydrogenase and aspartate transasminase (AST) are found in the mitochondria and cytosol.

41    DESCRIBE CONSTITUTIVE ENZYMES

These are enzymes that are synthesized in fixed amounts. The cells maintains these at a constant concentration.

42    DESCRIBE INDUCIBLE ENZYMES

These are enzymes that are synthesized as the cell needs them.  They are maintained in the cell at a rate required for life.  If the enzyme is not needed, it is not synthesized. If needed, the rate of synthesis is determined by the need.

43    DESCRIBE HOW THE LABORATORY CAN USE ENZYME TESTING TO LOCATE THE SITE OF TISSUE DAMAGE IN THE BODY AND LIST AN EXAMPLE

The three ways that the side of cellular or tissue damage can be determined is by:
A.    Assaying plasma or serum for organ specific enzymes such as creatine
        kinase (a muscle specific enzyme) or for glutamate dehydrogenase
        (which is liver specific).
B.    Assaying for the presence of isoenzymes.  Creatine kinase exists in three
        basic isoenzyme forms designated as MM, MB, and BB.   Ninety-five
        percent of this enzyme in a healthy individual exists in the MM form and
        may be due to is leakage during normal muscular activity.  If a patient is
        diagnosed with Duchenne muscular dystrophy, then the MB isoenzyme
        form will be significantly increased.
C.    Assaying for enzyme patterns in the serum and comparing the test results
        with known relationships of the enzymes in various organs or tissues.  For
        example, if ALT (alanine transaminase)  is measured and found to be in
        higher concentration than AST (aspartate transaminase), then that is a strong
        indication of liver damage.  Conversely, if AST is higher than ALT, then that
        would suggest that there has be myocardial damage.   Note, if an increase
        ALT is found along with an increase in the enzyme leucine aminopeptidase,
        then this not only indicates liver damage, but also cholestasis (bile duct
        blockage).

44    EXPLAIN BRIEFLY WHAT HAPPENS IN CARBON MONOXIDE POISONING

Carbon monoxide (CO) binds competitively at the same site on the Hgb molecule as does oxygen.  It binds 200 times stronger than oxygen and makes it difficult for oxygen to be transported to the tissues.

45    EXPLAIN WHAT IS MEANT BY THE BOHR EFFECT

When oxygen binds to the dimers of Hgb, there is a movement of L-tyrosyl residues and breaking of salt bonds. A proton is released with this breakage. There is another proton released from a COOH-terminal histidine residue. The freedom to dissociate increases with decreasing pH and the system tends to be acidic in the tissues. This results in the easy release of oxygen at lower pH values. This phenomenon is called the Bohr effect.

46    BRIEFLY DESCRIBE  CARBAMINOHEMOGLOBIN

About 10% of the carbon dioxide produced by body metabolism is transported bound to Hgb. The CO₂ that is covalently linked to Hgb (to NH₂-terminal valine amino acids) is called carbaminohemoglobin.

47    EXPLAIN THE CONCEPT OF COVALENT MODIFICATION USING PHOSPHOGLUCOMUTASE AS AN EXAMPLE

This is a set of enzyme reactions generally limited to the kinases and phosphatases. This is a control mechanism to alter enzyme activity in of the following three ways:
    a.     to make the enzyme less reactive.
    b.     to make the enzyme more reactive.
    c.     to change the direction in which the reaction goes.
   
This reaction uses the same enzyme (phosphoglucomutase). In the first reaction sequence, the enzyme acts as a phosphorylating enzyme (but the presence of a coenzyme is required) and in the second reaction step, it acts like a dephosphorylating enzyme. The enzyme is changing the way the direction the reaction goes. In most cases of phosphorylation, the enzyme changes a phosphorylated form to a non-phosphorylated form. In the case of the phosphoglucomutase enzyme, there is a serine residue present that can accept the phosphate group for transfer. This may be the events that are occurring:

Covalent modification enzymes are also found to act upon serine, threonine, and tyrosine amino acids. An example of this would be chymotrypsin:
a.     Chymotrypsin has a serine molecule at its 195 position. When chymotrypsin
        positions to cleave a peptide linkage, the serine molecule becomes the attacking
        nucleophile by forming a catalytic triad with aspartic acid (102 position) and
        histidine (57 position). Aspartic acid ionizes and pulls a proton from histidine
        which removes serine's hydroxyl group to form an a serine alkoxide which can
        attach the carbonyl residue of the peptide bond and break it. This activated
        enzyme is called an acylated enzyme.

48   NAME AND BRIEFLY DESCRIBE THE PHYSIOLOGICAL FEATURES OF AT LEAST TWELVE OF THE KNOWN BLOOD CLOTTING FACTORS

a.   Fibrinogen (I): is converted to fibrin. It consists of two tripeptide units. It is
      part of the intrinsic and extrinsic clotting systems. Has a biological half-life of
      up to 80 hours and is stable on storage.
b.  Prothrombin (II): is converted to thrombin which enzymatically attacks
     fibrinogen. It is part of intrinsic and extrinsic clotting systems. Has a biological
     half life of up to 70 hours and is stable on storage.
c.   Tissue thromboplastin (III): activates Factor X. Also called "tissue factor", it
      is part of the extrinsic clotting system. This is a tissue constituent comprised of
      protein and phospholipids and is found in most tissues.
d.   Calcium ion (IV): cofactor in several reaction steps. It is part of the intrinsic
       and extrinsic clotting systems.
e.   Proaccelerin (V): not an enzyme, but required for thrombin formation. It is
      part of the intrinsic and extrinsic clotting systems. It is also called "labile factor"
      and "prothrombin accelerator". Require calcium and phospholipids as cofactors.
      Although required for thrombin activation, it is activated by thrombin. Its
      biological half-life is up to 25 hours, is labile storage, and is heat labile.
f.   Accelerin (VI) is no longer included in coagulation tables. It was discovered to
      be an impure form of activated Factor V.
g.   Proconvertin (VII): activates Factor X. An endopeptidase and is part of the
      extrinsic clotting system. Also known as "Stable factor", "proconvertin", and
       "serum prothrombin conversion accelerator" (SPCA). It has a biological half-
       life of up to 5 hours. Requires calcium as a cofactor, stable in storage, and is
       heat labile.
h.   Antihemophilic globulin (AHG) (VIII): activates Factor X. A protein
       cofactor (required by another enzyme to function) and part of the intrinsic
       clotting system. Also called "antihemophilic factor" (AHF). Has a biological
       half-life of up to 16 hours and is labile on storage.
i.   Christmas Factor (IX): activates Factor VIII. And endopeptidase and part of
      the intrinsic clotting system. It is known as "antihemophilic factor B", and
      "plasma thromboplastin component (PTC). It requires calcium and
      phospholipids as cofactors. It has a biological half-life of up to 24 hours, is
      heat labile, and is stable on storage.
j.   Stuart Factor (X): activates prothrombin to thrombin. An endopeptidase and
     part of the intrinsic and intrinsic clotting systems. It is also called "Stuart-Prower
     factor". Requires calcium and phospholipids as cofactors. Has a biological half-
     life of up to 45 hours, is heat labile, and some stability during storage, and is
     heat labile.
k.   Plasma Thromboplastin Antecedent (PTA) (XI): activates Factor IX. An
      endopeptidase and part of the intrinsic clotting system. It is also known as
      "antihemophilic factor C". Requires calcium as a cofactor. Activates
      plasminogen to plasmin. Its biological half-life is about 60 hours and tends to be
       increased during storage.
l.   Hageman Factor (XII): activates Factor IX. An endopeptidase and part of the
      intrinsic clotting system. It has a biological half-life of 40 to 60 hours and is
      stable on storage.
m.  Transglutaminase (XIII): enhances cross-linking of fibrin. A transpeptidase
       and part of the intrinsic and extrinsic clotting systems. It is also called "fibrin
       stabilizing factor" (FSF). It has a biological half-life of up to 120 hours. It is
       stable in storage and is heat labile. It requires calcium to be activated.
o.   Other clotting factors to which a factor number has not been assigned are:
        [1]   Fletcher factor: Also called "prekallikrein". It is a serine protease,
                activating Factor XII and possibly Factor VII.
        [2]   Fitzgerald factor: Also called "Flaujeac factor" and "High Molecular
                Weight Kininogen or HMWK”. It is a cofactor in the activation of
                 Factors XI and XII.

49    DISCUSS THE ENZYME CASCADE MECHANISM USING THE COAGULATION OF BLOOD AS AN EXAMPLE

An enzyme cascade is a chain reaction of one enzyme acting on another. The coagulation of blood is considered to be the most complex of the cascade systems. A number of proteins, calcium, and phospholipids are required in the coagulation cascade. In the coagulation of blood, there are two systems that leads to this phenomenon; the intrinsic system and the extrinsic system.

Intrinsic System: This is the coagulation mechanism located inside the vascular system. It begins with the activation of Hageman factor (XII), which requires some type of trauma that ruptures and exposes collagen fibers in the vascular system. The intrinsic schematic is as follows:
   

Extrinsic system: The term extrinsic implies that the clotting process is initiated outside the vascular system. Tissue thromboplastin (III) is a membrane protein (outside the vascular system) that initiates the clotting process following injury. The other factor that is unique to the extrinsic system is proconvertin (VII) and binds to the Factor III in the presence of calcium. The extrinsic system is as follows:
                   
50    DISCUSS THE COMMON COAGULATION PATHWAY SCHEME AND SHOW WHERE THE EXTRINSIC AND INTRINSIC SYSTEMS INTERFACE WITH THE COMMON SYSTEM

The common pathway begins with the activation of factor X, followed by the conversion of prothrombin to its active form. Fibrinogen can be converted to a monomer which is followed by the cross-linking of the monomers to form a stable polymer. The following schematic illustrates the common pathway system.
               
51   LIST A MINIMUM OF FOUR LABORATORY TESTS THAT SCREEN FOR HEMOSTASIS DISORDERS AND DESCRIBE WHAT EACH TEST MEASURES

a.    Platelet count: identifies thrombocytopenia or thrombocytosis. Provides
        information, when combined with other clinical data, helps to identify platelet
        dysfunction.
b.    Prothrombin time (PT): measures factors (I, II, V, VII, X) of the intrinsic
       coagulation pathway. It detects vitamin K deficiencies, monitors oral anti-
       coagulation therapy, and detects the presence of inhibitors.
c.    Activated partial thromboplastin time (APTT): measures factors (I, II, V, VIII,
       IX, X, XI, XII, Fletcher, and Fitzgerald) of the intrinsic clotting system.
d.    Template bleeding time (TBT): is prolonged if platelet function is abnormal.
e.    Thrombin time (TT): is prolonged in hypofibrinogenemia, dysfibrinogenemia,
        heparin therapy, circulating Fibrin Degradation Products (FDP), pathological
        circulating inhibitors.
f.    Peripheral blood smear examination (diff): visual examination of platelets to
      evaluate normal or abnormal morphology (platelet dysfunction).

52    WHAT IS VITAMIN K AND EXPLAIN ITS ROLE IN COAGULATION

Vitamin K (fat soluble) is of two types: (1) Vitamin K₁ (phytylmenaquinone) found in green vegetables and (2) vitamin K₂ (multiprenylmenaquinone) synthesized by the intestinal bacteria.  It is required for the synthesis of clotting factors II, VII, IX, and X.  It is also required for the production of proteins C and S.  This vitamin carboxylates the glutamyl side-chains in the prothrombin molecule. The addition of these additional carboxyl groups increases the number of calcium binding sites and enhances the activation process by facilitating the binding of Ca⁺⁺ and phospholipids. See following schematic.
           
53    BRIEFLY DISCUSS THE ROLE OF COUMARIN ANTICOAGULANTS

These are analogs of Vitamin K and are known by the names of "dicoumarol", "bishydroxycoumarin", "warfarin", "panwarfin", and "warfilone". The administration of these drugs interferes with the biosynthesis of the clotting factors, especially prothrombin, leaving it deficient in calcium binding sites so that it does not enter the clotting mechanism.

54    BRIEFLY DESCRIBE HOW CALCIUM IONS ENTER INTO THE CLOTTING MECHANISM

Calcium ions forms complexes with any clotting factor that contains a carboxy-glutamate residue: prothrombin, II; proconvertin, VII; Christmas factor, IX; and Stuart factor, X.  Calcium is a ligand and it induces an "electric state" that permits interaction with other sequences in the clotting mechanism.  If calcium ions are absent or deficient, vitamin K dependent factors cannot bind with phospholipids and clotting is prevented.  Chelating agents as EDTA, citrate, or oxalate can be used as anticoagulants to remove calcium and prevent blood from clotting.  During a clotting event, platelets can release additional calcium to enhance hemostasis.

55    EXPLAIN THE ROLE OF PROTEIN C AND PROTEIN S IN HEMOSTASIS

These are proteins produced in the liver and require the presence of vitamin K.  Protein S is a cofactor to protein C and both are required to stop localized clotting initiated by activated factors V and VIII.  These two proteins will cause the degradation of these two activated factors. If one or both of these factors are deficient, a hypercoagulable state results.

56      RECOGNIZE AND/OR LIST THE FOLLOWING ACRONYMS

ALP        Alkaline phosphatase.  This is a generic designation as this enzyme is
                an isoenzyme that exists in five tissue-specific forms.
ALT        Alanine aminotransferase, formerly known as SGPT (serum glutamate-
                 pyurvate transaminase).  It has also been called glutamate-pyruvate
                 transaminase (GPT).
AST        Aspartate aminotransferase, formerly known as SGOT (serum glutamate-
                oxaloacetate transaminase).  It has also been called (oxaloactate
                transaminase (GOT).
CPK       Creatine phosphokinase.   Also referred to as CK (creatine kinase).
GGT        Gamma Glutamyltransferase.  It is also called glutamyl transpeptidase.
GLDH    Glutamate dehydrogenase.
ICD        Isocitrate acid dehydrogenase.  Present in liver, heart and skeletal
                muscle tissue.
LAP        Leukocyte Alkaline Phosphatase.   This refers to that isoenzyme found
                in leukocytes (specifically neutrophils and bands).
                A.    It is also the acronym for leucine aminopeptidase found in the
                        pancreas and liver tissues.  
LDH        Lactate Dehydrogenase.  Also referred to by the acronym LD.  Be
                 aware of the use of the LD acronym in a sentence as it can also
                 mean lethal dose
MDH      Malic acid dehydrogenase.     Found in cardiac, kidney, liver, and
                skeletal muscle tissues.

57    DESCRIBE ISOENZYMES

Isoenzymes, also called isozymes, are multiple forms of a related enzyme, but with different structures.  They vary in their physical, biochemical, antigenic, and immunological properties.  They may be multi-unit proteins.  They migrate to different Rf positions in electrophoresis.  These isoenzymes can catalyze the same reaction.

58    DISCUSS THE STRUCTURAL BASIS OF ISOENZYMES

Most encountered isoenzyme families are proteins composed of one or more polypeptide chains (subunits). If the subunits are the same in the primary, secondary or tertiary structure, they are called homopolymers. If the subunits differ, then they are called heteropolymers.
    ◊    A heteropolymer, consisting of two subunits ( A and B ) will have three
          possible enzyme compositions/structures.... AA, AB, and BB.
    ◊    IF the heteropolymer consists of three subunits ( A, and B ), there are four
          possible enzyme compositions/structures.... AAA, AAB, ABB, and BBB.
    ◊    IF the heteropolymer consists of four subunits ( A and B ), there are five
          possible enzyme conformations/structures.... AAAA, AAAB, AABB,
          ABBB, and BBBB .

59    EXPLAIN HOW ISOENZYMES MAY ORIGINATE

It is generally thought that isoenzymes are products of two related but separate genes.  Some of the amino acid sequences derived from these genes will be different and others the same.  It is estimated that about ⅓ or possibly more of the human enzymes are formed by one gene locus (site on the chromosome).  Many human enzymes exist in multiple form that are thought to arise from multiple gene loci.  Research studies show that the forms of a given enzyme may differ from one individual to another. It is conceded that different enzyme forms can arise from alleles.  An allele is an alternate form of genes located at the same loci.  If isoenzymes do originate from allelic genes, then these enzymes may be called allelozymes. (NOTE: The ABO blood groups arise from allelic genes.)  Enzymes known to arise from allelic genes are usually functional, but can have functional abnormalities and in some cases, have no enzymatic activity.

Isoenzymes to be discussed in this lesson [Lactic Acid Dehydrogenase (LDH) and Creatine Kinase (CK)] are known to be formed from subunits derived from different and distinct alleles. These isoenzymes may be described as hybrid enzymes. Review the following illustrations for forming these isoenzymes.
        structural gene "a" —> mRNA —> polypeptides —> enzyme subunit "O".
        structural gene "b" —> mRNA —> polypeptides —> enzyme subunit "☐".

If a dimer, as for creatine kinase, there are three possible combinations:
           
If a trimer, there are four possible combinations possible:
      
If a tetramer, as for LDH, then there are five possible combinations possible:
   
60    DISCUSS THE ISOENZYMES OF LACTIC ACID DEHYDROGENASE (LDH)

Also known as lactate dehydrogenase (LD), two gene produce similar, but non-identical polypeptides designated as "H", heart type isoenzyme and "M", muscle type isoenzyme. The LDH isoenzyme is a tetramer and can combine five different ways. LDH activity can be found in all cells of the body and tissue levels tend to be as much as 500 times higher than that found in serum. If tissues are damaged, LDH isoenzyme leaks into the body fluids, increasing serum levels. Examine the following table listing LDH isomer levels in human tissues.

            LDH levels in units per gram
                    Liver = 145
          Skeletal muscle = 147
          Heart = 124
          Erythrocyte = 36
          Kidney = 106

LDH, a tetrameric enzyme, combined five different way, is found in tissues in different types. The following table illustrates the type, composition, and where it is found in strongest concentration.

    Type           Composition                     Major Location
    LDH1         HHHH (H₄)               Myocardium, RBC's,
    LDH2         HHHM (H₃M₁)         Myocardium, RBC's, Kidney, Brain, Lymph
    LDH3         HHMM (H₂M₂)         Brain, Kidney, Lymphocytes
    LDH4         HMMM (H₁M₃)         Lung, Spleen, Neutrophils
    LDH5         MMMM (M₄)             Liver, Muscle, Skin, Neutrophils

LDH catalyzes the conversion of pyruvate to lactate with NAD⁺ as a hydrogen acceptor. The normal range for serum LDH ranges from 90 to 200 IU/L in most laboratories. There are some ranges with upper limits of 320 IU/L. After damage to a body tissue, the cellular breakdown release LDH and other enzymes into the blood stream. Measuring the tetrametric types of LDH determines which type of tissue was damaged. If a myocardial infarction occurred, then LDH serum values would exceed 200 UI/L and LDH₁ and LDH₂ type of isoenzyme will predominate.

61    DISCUSS THE ISOENZYME LDH IN MYOCARDIAL INFARCTION

When a myocardial infarction occurs, in most cases a 3 to 4-fold increase in serum LDH results. In some "infarct's" may result in a ten-fold increase over normal. If a myocardial infarct occurs, creatinine kinase (CK) will appear in the blood within 18 hours after the infarct. LDH will appear from 24 to 48 hours later. Examine the following graph comparing CK and LDH appearances.
          

62    BRIEFLY DISCUSS THE ISOENZYME LDH IN HEMOLYSIS

If hemolysis is severe, the LDH pattern resembles that of a myocardial infarction. Isoenzymes are of the LDH₁ type.

63    BRIEFLY DISCUSS THE ISOENZYME LDH IN MEGALOBLASTIC ANEMIA

RBC's hemolyze and release LDH₁ and LDH₂ type isoenzymes. The elevations may be 50 times that of normaL.

64    BRIEFLY DISCUSS THE ISOENZYME LDH IN LIVER DISEASE

Total LDH may double in cirrhosis and obstructive jaundice.  Primary liver disease or liver anoxia is characterized by an increase in the LDH₅ type isoenzyme.

65    BRIEFLY DISCUSS THE ISOENZYME LDH IN MALIGNANT DISEASE

There is an increase in total LDH in the serum with LDH₄ and LDH₅ demonstrating the greatest increases in malignant disorders. If there is a germ cell tumor (of the ovary or testes), then LDH₁ is the most elevated isoenzyme.

66    BRIEFLY DISCUSS THE ISOENZYME LDH IN PROGRESSIVE MUSCULAR DYSTROPHY

LDH increases are of the LDH₅ type during the early and middle stages of the disease. As muscular dystrophy progresses into the late stages, the LDH type shifts to LDH₁ and LDH₂ type isoenzymes.

67    BRIEFLY DISCUSS THE ISOENZYME LDH IN LYMPHOID TUMORS

There are three types of LDH isoenzymes present; LDH₂, LDH₃, and LDH₄. LDH₂ and LDH₃ will be the predominate forms present in lymphoid tumors.

68    DISCUSS THE ISOENZYMES OF CREATINE KINASE (CK)

Creatine kinase is the predominate isoenzyme of skeletal muscle. It is formed of peptide subunits designated as M (type found in muscle) and B (type found in the brain). CK subtypes are MM (CK₃), MB (CK₂), and BB (CK₁). CK₁ is found in brain, colon, bladder, placenta, prostate, lung, uterus, and thyroid tissues. CK₂ is found predominately in heart muscle and to some extent in skeletal muscle. Trace amounts are found in other tissues. CK₃ is found in skeletal muscle and heart muscle. It is in higher concentration in heart muscle than CK2. CK2 may make up 42% of total CK activity. Creatine kinase catalyzes the following reaction:
 
Like LDH, it increases in blood when there is cell damage.  Measuring the type of CK dimers determines the type of damage.

69    DISCUSS CREATINE KINASE IN HEART DISEASE

Total creatine kinase (CK) and CK₂ are considered to be more important than most enzyme tests in the diagnosis of myocardial infarctions. CK₂ increases rapidly and peaks in about 24 hours after the infarct. CK₂ has a short half-life and will return to normal levels by day three. CK₃ increases and peaks about 12 hours after CK₂. CK₃ has a longer half-life and usually returns to normal by day four. Creatine kinase increases in cardiac surgery or any other type of heart tissue trauma. CK is not affected by tachycardia, angina pectoris, or congestive heart failure.

70    DISCUSS CREATINE KINASE IN MUSCLE DISEASE

Creatine kinase is greatly increased in all types of muscular dystrophy and most all other forms of muscular disorders. CK is normal in myasthenia gravis, multiple sclerosis, poliomyelitis, and Parkinson's disease.  If chronic alcoholism is in those patients diagnosed with muscular myopathy or neuropathy, then CK may be increased.

71    LIST A MINIMUM OF FOUR CENTRAL NERVOUS SYSTEM DISORDERS IN WHICH CREATINE KINASE IS ELEVATED

(1) cerebral ischemia, (2) acute cardiovascular disease, (3) head trauma, (4) Reye's syndrome (with brain swelling characterized by fatty infiltration and liver dysfunction).

72    STATE WHETHER CREATINE KINASE (CK) IS NORMAL OR ABNORMAL IN THYROID DISEASES

In hypothyroid disorders, CK is increased, whereas in hyperthyroid disorders CK is decreased.

73    LIST SEVEN INHIBITORS OF CREATINE KINASE

(1) manganese ions, (2) zinc ions, (3) copper ions, (4) calcium ions, (5) any sulfhydryl binding reagent, as iodoacetate, (6) excess ADP, and (7) cystine

74   DISCUSS THE ISOENZYMES OF ALKALINE PHOSPHATASE (ALP)

Alkaline phosphatase (ALP) is an ubiquitous enzyme that is elevated in bone and liver diseases. Mg⁺⁺, Co⁺⁺, and/or Mn⁺⁺ are required to activate this enzyme. Zn⁺⁺ is required as a constituent ion metal ion. It is inhibited by phosphate, borate, oxalate, and cyanide ions. ALP is found in high levels in intestinal epithelium, kidney tubules, osteoblasts of bone, liver, placenta, adrenal glands, prostate gland, leukocytes, and erythrocytes. It has an important role in the calcification of bone. There are five tissue-specific isoenzyme forms known.  At one time it was thought that there is no clinical advantage to an isoenzyme analysis of ALP. When testing for ALP, it is usually for total ALP.  Specific ALP testing does take place to determine if bone or liver tissue is the source of increase enzyme activity.  This test has been shown to be useful in the diagnosis of (1) hepatobiliary disease, (2) bone disease, (3) Down's syndrome, (4) polycythemia vera, (5) sickle cell anemia, (6) and proximal nocturnal hematuria.  Fractionation of ALP has also been used to monitor Paget's disease, osteoporosis, and bone cancer.

75    EXPLAIN HOW LEUKOCYTE ALKALINE PHOSPHATASE IS RELATED TO ALP

ALP is present in leukocytes as Leukocyte Alkaline Phosphatase (LAP). It is one of the enzymes of the tertiary granules of neutrophils, bands, and some metamyelocytes. The more matured the granulocyte, the more LAP is present in the granules. This feature is used to differentiate a number of hematologic disorders. Review the following table to note how LAP concentrations vary in seven hematological disorders assuming that a normal LAP value is from 11 to 95. Maximum LAP score is 400. (Note: Normal LAP values may vary from lab to lab, example:  13 - 100).

        Leukemoid reaction increased ( >95 )
        Chronic granulocytic leukemia decreased ( <11 )
        Acute granulocytic leukemia variable
        Polycythemia vera increased ( >95 )
        Myelofibrosis     (variable)
        Proximal Nocturnal Hematuria (PNH) decreased ( <11 )
        Pregnancy (third trimester) increased ( >95 )

76   BRIEFLY DESCRIBE TROPONIN AS A CARDIAC MARKER

Troponin (the general name) is a cardiac specific molecule comprised of three subunits, a complex molecule found on the actin filament of muscle.  These proteins are present in myocardial and skeletal muscle cells, but not in smooth muscle.  It consists of (1) troponin T, designated as TnT; (2) troponin C designated as TnC, and (3) troponin I, designated as TnI. These three subunits are not strucutrally or genetically related.   These three proteins function to regulate muscle contraction.  As a test for the presence of myocardial infarction, it is deemed to be equivalent to CK-MB or better.  The troponin markers appear and rise parallel to the isoenzyme CK-2, but there will be a greater increase that CK-2.   These proteins are superior to LDH isoenzymes as a test for acute myocardial infarction (AMI).

77    EXPLAIN THE EVENTS THAT ALLOW TROPONIN TO APPEAR IN THE BLOOD

When ischemia of the myocardium occurs, it is followed by reperfusion (return of the blood supply back into the damaged area of the myocardial infarction). As reperfusion takes place, the degradation of the troponin is spontaneous in the damaged myocardial muscle fibrils and is “washed” from the cytosol of the damaged heart cell into the blood stream.

78    BRIEFLY DISCUSS TROPONIN I AND TROPONIN T AS CARDIAC MARKERS

Both Troponin I and Troponin T appear in the blood about the same time and both provide similar information.  Troponin T will appear in significant detectable levels in 3 to 4 hours after the infarct and will remain elevated for ten to fourteen days. Troponin I is considered by some primary care providers to be a more specific and better cardiac marker that TnT.   Both will peak in 14 to 20 hours and return to normal levels over a 5 to 10 day period.  Neither of these proteins normally circulate in the blood.   Troponin I is 12-13 times more abundant in myocardial tissue that CK-2.  Small amounts of cardiac necrosis will release measurable amounts of troponin into the blood.  TnI can be used as an indicator of the morbidity and mortality of patients diagnosed with ischemic heart disease. 

79   DEFINE MYOSIN

Myosin is a contractile protein that makes up about 54% of striated muscle. These are the thick filaments of the sarcomere.

80    BRIEFLY DESCRIBE ACTIN

Actin is a contractile protein making up about 25% of striated muscle. These are the thin filaments of the sacromere. Resting actin exists as G-actin, but is converted to F-actin when bound to Ca⁺⁺ and interacts with ATP. F-actin is a highly polymerized, supercoiled α-helix that enters into the contraction process. It is a complex molecule and contains tropomyosin and troponin, which act to inhibit ATPase activity of the myosin-actin complex and blocks the interactino of actin and myosin molecules. For every seven moles of actin, there is one mole of tropomyosin, troponinT, troponin C, and troponin I each. Examine the following illustration of the actin and troponin complex.
   

81    DESCRIBE  TROPOMYOSIN

Tropomyosin is a double α-helical protein unit (dimer) that winds about each other and is located in the strands of the actin molecule. It consists of elongated dimers that exist the length of the actin molecule. It is bound with the troponin complex (troponin-T, troponin-I, troponin-C).

82    BRIFELY DISCUSS THE FUNCTION OF TROPONIN-C

Troponin-C is the Ca⁺⁺ binding subunit. When the calcium ion binding sites are occupied, this blocks the inhibition effects of troponin-I and allow the actin-myosin interaction to take place. How this process works is not fully understood.

83     BRIEFLY DESCRIBE THE FUNCTION OF TROPONIN-T

Troponin-T is the subunit that binds the troponin complex to the tropomyosin molecule.

84    BRIFELY DESCRIBE THE FUNCTION OF TROPONIN-I

Troponin-I functions in concert with tropomyosin to inhibit the binding of actin to myosin and prevent contraction of the sarcomere. As long as Ca⁺⁺ concentration does not exceed the amount needed for contraction, the muscle fibers are in the resting state. A nerve impulse will cause the release of sufficient Ca⁺⁺ to “override” the inhibitory mechanism and allow contraction to occur.

85    DISCUSS THE ENZYME ASPARTATE TRANSAMINASE (AST)

Formally known as serum glutamate oxaloacetate transaminase (SGOT or GOT), it is also known as L-aspartate:2-oxoglutarate aminotransferase (AST). It catalyzes the intraconversions of amino acids and α-oxoacids by transfering amino groups. An example is shown:
   
 

This is an isoenzyme widely found in the tissues of the body and is normally present in the bile, cerebrospinal fluid, plasma, and saliva. Normal plasma values for the adult range from 8 to 20 IU/L. Newborn and infant values are higher, up to 75 IU/L. One isoenzyme form is found in the cytoplasm of the cell and another is found in the mitochondria. Clinically, this enzyme will elevate in viral hepatitis and other liver disorders (with necrosis) before clinical signs/symptoms are evident. It is a better indicator of liver disfunction. Elevations from 20 to 50 times that of normal is the rule, but may be 100 fold in some patients. It takes from seven to twelve days to reach peak elevated values and it fades to normal values in three to five weeks. It is less specific for liver than alanine transferase (ALT), which is a more liver specific enzyme and elevations of ALT persist longer than AST.

The AST level will elevate following a myocardial infarction, usually increasing six to eight hours after the onset of chest pain and peaks at about 24 hours, returning to its pre-infarction level about the fifth day. It is not cardiac specific and is a poor indicator of a cardiac infarct. AST is also elevated in diseases of the lungs and skeletal muscle. Aspartate aminotransaminase levels may increase two to three times over normal for pulmonary emboli but levels will increase from two to five times that of normal for acute pancreatitis, crushed muscle injuries, gangrene, and hemolytic disease. AST levels do not increase significantly in progressive muscular dystrophy and dermatomyositis.

86    DISCUSS THE ENZYME ALANINE TRANSAMINASE (ALT)

Formally known as serum glutamate pyruvate transaminase (SGPT or GPT), it is also known as L-alanine:2:oxoglutarate aminotransaminase (ALT).

ALT is found in bile, cerebrospinal fluid, plasma, and saliva. It is also found in other tissues. ALT levels are elevated in viral hepatitis and liver disorders with necrosis (even before clinical signs and symptoms appear). In most patients, this enzyme will elevate from 20 to 50 times that of normal, but in some cases may be elevated 100 fold. This enzyme will peak between the seventh and twelfth days and return to normal values in three to five weeks. ALT values tend to be higher than AST values in liver disorders since it is a more liver specific enzyme. It will also remain elevated longer than AST.

In a myocardial infarction, alanine transaminase may slightly elevate, but as a rule it will remain within the normal range. Normal range for the adult is 8 to 20 IU/L. Newborn and infant values may be as high as 75 IU/L.

ALT values are increased up to eight times that of normal in progressive muscular dystrophy and dermatomyositis, but is not responsive in muscular diseases of neurogenic origin.

87   DISCUSS THE ENZYME GLUTAMATE DEHYDROGENASE

Also known as L-glutamate:NAD(P) + Oxidoreductase, this enzyme is a mitochondrial enzyme that requires the cofactor Zn⁺⁺.  and is a seldom used test in the typical laboratory. This enzyme is present in trace amount in human plasma, but is increased in liver disease with hepatocellular damage. It is elevated in response to hepatotoxic agents and also in metastatic diseases involving the liver. This enzyme may be used to evaluate glutamate and ammonium balance in the cell.  The enzyme removes hydrogen from glutamate to form an imino acid.  Testing for this enzyme is located primarily in specialty laboratories for diagnostic enzymology.

Note:  The prefix 'imino' denotes a '=NH' group or a disubstituted nitrogen in amines.

88    USING ACUTE HEPATITIS AS AN EXAMPLE, DESCRIBE HOW ALT, AST, AND GLDH MAY BE USED TO FOLLOW ENZYME CHANGES IN A DISEASE

In the disease acute hepatitis, there are four phases the disease progresses through before the patient may be designated as cured.   The first phase is the incubation period in which cellular changes are taking place and the serum values of ALT (alanine aminotransaminase), AST (aspartate aminotransferase), and GLDH (glutamine dehydrogenase) remain within the normal range.  During the first 1.5 weeks of this incubation phase there is little change in the serum enzyme levels.   In the second or prodome phase, there is an increase in the ALT  and AST enzymes over period of approximately 10 days.  The AST  level of transaminase will usually rise higher than the ALT.   During the prodrome phase the GLDH will increase slightly and fall back to low values over a 2 week period.  The ALT and AST abnormal levels will continue for a 3 to 4 week period and then return to normal between the third or icteric phase and the fourth or convalescence phase.  During the period of time in which there are elevated ALT and AST levels, hepatocellular damage has occurred and there is a corresponding depression of the liver's ability to conjugate bilirubin.   Bilirubin levels will rise and peak along with the ALT and AST levels.  As the patient progresses through the icteric phase and enters the convalescence phase, the ALT, AST, and bilirubin values return to normal.  If during the convalesence period, the patient becomes physically active, serum increases in ALT can be observed.  Refer to the following illustration demonstrating the rise and fall of these enzyme and how the ability of the liver to conjugate bilirubin decreases during the disease phase.

                                Enzyme changes in acute hepatitis.

89    EXPLAIN WHY A PHYSICIAN ORDER ENZYME ASSAYS TO FOLLOW THE PROGRESS OF CHRONIC LIVER DISEASE

Enzyme activity changes more quickly to changes in liver function than any other parameter.  By following the changes that occur in levels of enzyme activities, the course and progress of the disease can be followed.  If there is an acute necrosis involving the hepatocytes, it is possible to recognize significant detrimental changes by looking at the transaminase enzymes.    For example, if the patient is diagnosed with chronic hepatitis and there are repeated episodes of acute attacks, AST tends to increase over the long term whereas the ALT tends to decrease.  If the patient has an active cirrhosis of the liver that is not characterized by acute attacks, then the ALT tends to remain elevated over the AST.   It has been found in patient (who have an alcoholism problem), but with no recognizable hepatocyte damage, the AST tends to be slightly elevated over the ALT, yet both transaminases are only slightly elevated above the normal range.

Note that the use of the term active in chronic liver disorders just indicate that hepatocyte damage is taking place.

90    LIST EXAMPLES OF PLASMA-SPECIFIC ENZYMES

Any of the serine protease procoagulants.  Examples are thrombin, Hageman factor, Stuart factor, fibrinolytic enzymes as plasminogen and plasminogen activator.

91    LIST EXAMPLES OF SECRETED ENZYMES

Lipase, α-amylase, trypsinogen, cholinesterase, and prostatic acid phosphatase.

92    LIST EXAMPLES OF CELLULAR ENZYMES

Lactate dehydrogenase, aminotransaminases, and alkaline phosphatases.

93   DISCUSS THE ENZYME GAMMA-GLUTAMYLTRANSFERASE (GGT)

This enzyme is also called 5-glutamyl-peptide:aminoacid-5-Glutamyltransferase (GGT).  GGT is a peptidase that transfers an amino acid to a peptide or a peptide to an amino acid, hence an amino acid transferase. This enzyme requires that the peptide contain a terminal glutamate residue.  GGT is present in serum and in all cells except muscle cells.  It is cell membrane enzyme.  GGT is elevated in all forms of liver diseases, with its elevation being the highest in intra- or post-hepatic biliary obstruction.  It is considered to be the best indicator for hepatobiliary disorders.  It is of little value in differentiating between different types of liver problems.

Elevated levels of γ-glutamyltransferase are observed in [1] fatty livers, [2] acute and chronic pancreatitis, [3] certain pancreatic malignancies, [4] infectious hepatitis, [5] drug intoxication, [6] alcoholism, [7] alcoholic cirrhosis, [8] cystic fibrosis with hepatic complications, and [9] prostatic cancer. If an elevated GGT is observed in a patient diagnosed with a malignant disorder, it is likely to be metastatic to the liver. Normal values for the adult male is 9 to 50 IU/L and for the adult female, 8 to 40 IU/L.

94    DISCUSS THE ENZYME AMYLASE

Alpha amylase is a human hydrolase that splits complex carbohydrates composed of α-D-glucose units linked by a α-1,4-linkage (such as starch, amylose, amylopectin, and glycogen). α-amylase is an endo-amylase that attacks the carbohydrate internally and randomly. The end products of this type of cleavage are limit dextrans, maltose, and glucose. This enzyme cannot attack glucose molecules attached by an α-1,6-linkage. This enzyme is also designated as a metalloenzyme, requiring Ca⁺⁺ to function. Amylase is a small molecular weight molecule and is excreted by the pancreas and salivary glands. This enzyme will migrate electrophoretically with the β and γ immunoglobulins. This enzyme (designated as P-amylase) is present in several organs and tissues, but is produced principally by the acinar cells of the pancreas. The salivary gland also produce a S-type amylase which is destroyed by the acidity of the stomach. Amylase activity can be demonstrated in [1] adipose tissue, [2] lungs, [3] ovaries, [4] oviducts, [5] semen, [6] striated muscle, and [8] testes. Amylase consists of three isoenzymes, but current testing strategies does not differentiate between the isoenzyme types.

Clinically, the testing of serum for amylase, is usually for the diagnosis of acute pancreatitis. The serum levels will rise within two to twelve hours and peak between 12 and 48 hours. Normal levels are attained in three to four days. Amylase values may be helpful in confirming the diagnosis of ascites, pleural effusions, and pseudocysts, Acute pancreatitis may be difficult to confirm with the amylase test since other disorders such as perforated gastric ulcer, perforated duodenal ulcer, intestinal obstruction, or mesenteric vascular obstruction can be characterized by hyperamylasemia. Normal values may vary by the procedure used. The Beckman DS method reports a normal range of 25 to 125 IU/L for serum and 1 - 17 IU/L for urine.

Beta amylase is of plant and bacterial origin that cleaves the complex carbohydrate from the terminal end.

95    DISCUSS THE ENZYME LIPASE

Lipases, also designated as triacylglycerol acylhydrolases are enzymes that hydrolyze glycerol esters of long chain fatty acids, selectively attacking the ester bonds at the α and α1 carbon positions.  The products of this reaction are β-monoglycerol (2-acylglycerol) and fatty acids. Most lipase is produced in the pancreas, but some is secreted by the tongue and the mucosa of the stomach, intestine, and pulmonary system.  Lipase activity can be demonstrated in leukocytes and adipose cells. Lipase testing is to diagnose pancreatic disorders. If an acute pancreatitis is suspect, the lipase values will significantly rise in two to twelve hours (2 to 4 times normal), peak and drop back to normal levels in the next 48 to 72 hours.  Elevated values have been reported to exist for up to ten days.  Lipase testing may be helpful in diagnosing acute pancreatitis, chronic pancreatitis, obstruction of the pancreatic duct, perforated gastric ulcer, perforated duodenal ulcer, intestinal obstruction, and mesenteric vascular obstruction.  Normal values (BMD turbidimetric method) are from 10 to 150 IU/L for the average adult.  Adults over 60 may demonstrate a normal range of 18 to 180 IU/L.

96    DISCUSS THE ENZYME TRYPSIN

Trypsin is a serine proteinase that attacts peptide bonds formed by carboxyl groups of lysine or arginine with any of the amino acids. There are two different forms of trypsin (trypsin I and trypsin II).  Trypsin activity is dependent upon calcium, magnesium, manganese, and cobalt ions. Citrate, cyanide, fluoride, sulfide, and heavy metals (Hg, Pb) exert an inhibitory effect on the enzyme. Testing for trypsin should be performed upon fresh aspirated specimens of the duodenum.  Fresh feces may be used, but if the stool transits the intestine slowly, then pancreas trypsin may be destroyed by proteases.  Trypsin testing is helpful in the diagnosis of chronic pancreas and cystic fibrosis. Normal values, using synthetic peptide substrates, for both children and adults ranges from 40 to 760 μg trypsin/gram of stool specimen.

97   DISCUSS THE ENZYME CHOLINESTERASE

Also called acetylcholine acetylhydrolase, this enzyme exists in two forms.
    A.     True cholinesterase or cholinesterase I is found in RBC’s, lung, spleen,
              nerve endings, and gray brain matter.
    B.     Pseudocholinesterase (also known as acycholine acylhydrolase, benzoyl
             cholinesterase, or cholinesterase II) is found in the heart, liver, pancreas,
             and white brain matter.  It is the assay of this enzyme that is clinically
             useful because it falls more rapidly in adverse situations.
Cholinesterase testing is useful as an indicator for organic phosphorus toxicity (insectide poisoning). It may be useful in testing for liver function. Cholinesterase decreases in acute hepatitis, chronic hepatitis, and carcinoma with metastasis to the liver. Normal values are the rule in chronic hepatitis, mild cirrhosis of the liver, and obstructive jaundice. Normal values are determined by the testing method used. Dibucaine inhibition causes a 82.3% to 84.9 % inhibition. The fluroride inhibition technique causes a 78.5% to 80.9% inhibition.

98    DISCUSS THE ENZYME ACID PHOSPHATASE (ACP)

Also known as phosphohydrolase (ACP)or orthophosphoric-monoester, this enzyme in present in all cells, because of their location in the lysosome.  Acid phosphatase is a generic term that describes all of the forms of enzyme.  Acid phosphatase is present in the greatest concentration in bone marrow, erythrocytes, liver, milk, platelets, spleen, and prostate gland. Acid phosphatase is found in highest concentrations in the prostate gland, perhaps because the pH = 5.0 to 6.0.  Osteoclasts also produce acid phosphatase.  ACP is unstable at temperatures above 37 0C and pH levels > 7.0.  If a blood specimen is collected for acid phosphatase testing, be alert to the fact that if the blood sample is allow to sit at room temperature for one hour, over 50% of its activity may be lost.  If the specimen is collected in citrate, the blood pH will be stabilized around 6.5 and preserving the enzyme.

The greatest value in testing for acid phosphatase concentrations is the diagnosis and monitoring of prostate cancer.  Prostate-type acid phosphatase is different from other tissue-type acid phosphatases.  Testing strategies employ formaldehyde and cupric ions which inhibits erythrocyte-type acid phosphatase, but not prostate-type.  Blood collected for ACP testing must be hemolysis free and the serum separated ASAP. Normal acid phosphatase values (via RIA) are less than 3.0 ng/mL.  Clinical values reported in males with prostate cancer with metastases may be 40 to 50 times that of normal.  Total non-prostatic ACP activity increases in Paget’s disease, hypoparathyroidism, malignant cancer invasion of bone tissue.  Non-prostatic ACP activity is increased in Gaucher’s disease, Niemann-Pick’s disease, myelocytic leukemia, and certain hematological disorders.

Because semen is contains high concentrations of ACP, this fact is used in forensic medicine to investigate rape and other sexual offenses.

99    DISCUSS THE ENZYME TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE

Terminal deoxynucleotidyl transferase (TdT) or DNA Nucleotidylexotransferase is a DNA polymerase. This is a less frequent used test assay because it is restricted to thymocytes and precursor cells of bone marrow lymphocytes. Its value lies in the differentiation of lymphocytes. It helps to diagnose T-cell lymphoblastic leukemias and lymphomas. The highest concentrations of TdT demonstrated in lymphocytes is in acute lymphoblastic leukemia. Other disorders in which elevated TdT can be demonstrated are chronic myelogenous leukemia (30% cases) and acute undifferentiated leukemia (50% cases). Caution is required in interpreting leukemic abnormalities in children in that up to 10% of nucleated bone marrow cells may contain TdT. Elevated terminal deoxynucleotidyl transferase may be observed in marrow regeneration, idiopathic thrombocytopenic purpura, and neuroblastoma. Testing for TdT may be accomplished through radioactivity testing, immunofluorescent methodology, or Enzyme-Linked ImmunoabSorbent Assay technology.

100    DISCUSS THE ENZYME ISOCITRATE DEHYDROGENASE (ICD)

Also known as Isocitrate:NADP+ Oxidoreductase (Decarboxylating), this is a rarely requested enzyme assay. The enzyme is substrate specific and is a sensitive indicator of parenchymal liver disease. It can detect early hepatocyte malfunction. Highest values are observed in viral hepatitis. Aspartate aminotransferase and alanine aminotransferase provides basically same useful clinical informaiton as that of the isocitrate dehydrogenase test.

101    EXPLAIN HOW LEAD EXERTS ITS NON-COMPETITIVE INHIBITION EFFECT UPON ENZYMES

Lead forms covalent bonds with the free sulfhydryl groups of cysteine. This bonding has the effect of denaturing the enzyme and once bonded, the covalent complex is irreversible. Since enzymes tend to be present in the cell in small amounts, it does not require much lead to produce pronounced metabolic effects. When lead inactivates just one key enzyme, toxic effects are manifested. The heme biosynthesis system is sensitive to lead poisoning and produces some of the first symptoms. Lead affects the enzyme ferrochelatase (required to insert Fe⁺⁺ into the heme structure, protoporphyrin IX). Lead covalently bonded with ferrochelatase results in the accumulation of aminolevulinate, a corresponding buildup of porphyrin in the tissues, and anemia.

Note.  Many enzymes depend upon sulfhydryl groups for activity. Such sulfhydryl groups are capable of forming tight covalent bonds with many of the heavy metals. Lead, mercury, and silver are especially toxic. Interestingly, if iron and copper are present in the tissues in large amounts, they can cause intoxication with enzyme inactivation.

102    LIST NINE COMMONLY ASSAYED ENZYMES AND STATE THEIR CLINICAL USEFULNESS

            Enzyme                                 Clinically useful to detect
[1]   Acid Phosphatase                       prostatic carcinoma
[2]   Alkaline Phosphatase                 liver or bone disease
[3]   Amylase                                        pancreatic diseases
[4]   Glutamate Aminotransferase     liver or heart disease
[5]   Aspartate Aminotransferase       liver or heart disease
[6]   Alanine Aminotransferase          liver or heart disease
[7]   Lactate Dehydrogenase              liver or heart or RBC disease
[8]   Creatine Kinase                          heart or muscle or brain disease
[9]   Troponin                                     heart disease

103    LIST THIRTEEN LESS COMMONLY ASSAYED ENZYMES THAT HAVE SOME DEGREE OF CLINICAL USEFULNESS

          Enzyme                                                            Clinically useful to detect
[1]    Aldolase                                                            muscle or heart disease
[2]    Ceruloplasmin                                                 liver disease (Wilson’s disease)
[3]    Elastase                                                             collegen diseases
[4]   Glucose-6-Phosphate Dehydrogenase             erythrocytes
[5]    γ-Glutamyl Transpeptidase                             liver disease
[6]   Glutathione Reductase                                    anemia or cyanosis
[7]   Hexose 1-Phosphate-Uridyl Transferase        galactosemia
[8]   Lipoprotein Lipase                                          hyperlipoproteinemia
[9]   Ornithine Transcarbamylase                          liver disease
[10]   Pepsin                                                             stomach disorders
[11]   Plasmin                                                           blood-clotting disorders
[12]   Pseudocholinesterase                                     insecticide posioning, hepatic
                                                                                     toxins
[13]   Trypsin                                                            pancreas and intestine disorders
[14]   Cholinesterase*                                              chronic hepatitis dysfunction,
                                                                                   scoline sensitivity

* There are four cholinesterase variants.  If a patient is administered scoline (a muscle relaxer) used in anesthesia and the patient has abnormal cholinesterase activity, respiratory paralysis occurs. This test is requested to detect the variants.  It is estimated that 0.05% of the people are at risk.

104    DESCRIBE THE STORAGE POTENTIAL OF SELECTED ENZYMES

Enzyme                    Temperature (^OC)        Time in Storage
Aldolase                           22-24                           up to 2 days
                                            0 to 4                         up to 2 days
                                             ―25                unstable as it deteriorate on thawing
Alanine                              22-24                          up to 2 days
   Aminotransferase           0 to 4                        up to 5 days
                                           ―25                  unstable as it deteriorates on thawing
α- Amylase                     22-24                            up to 1 month
                                            0 to 4                           up to 7 months
                                           ―25                              up to 2 months
Aspartate                        22-24                             up to 3 days     
   Aminotransferase        0 to 4                            up to 7 days
                                            ―25                             up to 1 month 
Ceruloplasmin                  22-24                           up to 1 day
     (Ferroxidase)              0 to 4                            up to  14 days
                                             ―25                            up to 14 days
Cholinesterase (CHS)      22-24                           up to 7  days
                                           0 to 4                             up to 7 days
                                              ―25                            up to 7 days
Creatine Kinase (CK)      22-24                            up to 7 days
                                           0 to 4                             up to 7 days
                                             ―25                              up to 1 month
Lipase  (LPS)                   22-24                             up to 7 days
                                           0 to 4                              up to 21 days
                                             ―25                              up to 21 days
Phosphatase  (ALP)           22-24                up to 3 days (activity may increase)
     Alkaline                        0 to 25                           up to 3 days
                                              ―25                              up to 1 month
Note that 22 - 24 ^OC represents room temperature

The stability of acid phosphatase (ACP) is determine if is acidified or not.  At
22-24 ^OC, it is stable for about 4 hours if unacidified.  If it is acidified with
acetate or citrate, it is stable for up to 3 days at 0 to 4 ^OC and has the same
stability if frozen at ―25 ^OC.

This web site is maintained by Whitney Williams, wwilliam@astate.edu

This page last updated 07/28/08