Enzymes - Clinical Tree

Diagnostic Enzymology

The principle of diagnostic enzymology is that various disease conditions cause increased cell membrane permeability to macromolecules or outright lysis of the cell membrane that allow macromolecules, dissolved in the cytoplasm, to leak into the extracellular space and gain access to the bloodstream. This permits assay of enzyme activity directly (including immune reaction) that would identify abnormally high levels in blood. Some of these enzymes have multiple forms, based on their content of different subunits, where certain forms predominate in specific tissues, making it possible to identify the tissue source of the damage. Invariably, a combination of measurements will identify the tissue source of the elevated enzyme activity. Abnormally released enzymatic activities demonstrate a pattern of release over time after the disease event (tissue damage, cell death, hypoxia, infection, or inflammation) that may be characteristic of the diseased organ in addition to the activity of the released enzyme itself. Moreover, the healing process can be reflected in the course of measurements, and sometimes, prognosis of a disease condition can be reflected by changes in the released activity. The activity of the released enzyme is usually proportionate to the extent of tissue damage and must be sizeable enough to withstand the dilution by the general circulation (about eight quarts). The activities of certain enzymes are of interest in diagnosing certain diseased tissues ( Table 5.1 ).

Table 5.1

Certain Enzymatic Activities are Useful in Determining the Organ From Which They are Derived During a Disease Process.

Tissue	Useful Enzyme Activity in Serum
Heart, liver, muscle	Lactate dehydrogenase
Muscle, also cardiac muscle	Creatine kinase
Liver	Glutamyl transferase
Heart and liver	Alanine aminotransferase
	Glutamate-pyruvate transaminase
	Aspartate aminotransferase
	Glutamate-oxaloacetate transaminase
Pancreas	α-Amylase
Prostate	Acid phosphatase (tartrate labile)
Bone, intestine (others)	Alkaline phosphatase (ALP)

Note: Another abbreviation used for alanine aminotransferase (AAT) is ALT (alanine transaminase).

Enzymes With Multiple Subunits: Tissue-Specific Isozymes

Enzymes, if they happen to have multiple subunits, can be extremely useful in diagnosis. A case in point is lactate (or lactic acid) dehydrogenase (LDH) . These isoenzymes can be separated by gel electrophoresis. Fig. 5.1 shows the tetrameric LDH. It consists of four subunits composed to two separate kinds, the heart-type subunit (H) and the muscle-type subunit (M).

Figure 5.1, Lactate dehydrogenase (LDH) catalyzes the reaction shown at the top of the figure. The models in gray or yellow show the composition of the dimers and tetramers. The H 4 (LDH1) enzyme reflects the heart myocardium and erythrocyte, and the M 4 (LDH5) reflects liver and muscle primarily.

A gel electrophoretic separation of these isoenzymes in normal serum is shown in Fig. 5.2 .

Figure 5.2, Separation of LDH isozymes by gel electrophoresis. The uppermost spot on the left is LDH1 (H 4 ) and, in descending order: LDH2 (H 3 M 1 ), LDH3 (H 2 M 2 ), LDH4 (H 1 M 3 ), and LDH5 (M 4 ). Note that in normal serum, LDH2 is greatest in quantity followed by LDH1 as shown in the table on the right. The tracings on the left, indicating the relative amounts arise from densitometry measurements of the stained enzymes.

Specific staining of the electrophoretic spots of LDH isozymes involves the enzymatic assay on the gel by the following method: lactate and NAD+ are added to form pyruvate+NADH+H+. To react with the NADH, phenazine methosulfate is added to form phenazine methosulfate+ tetranitro blue tetrazolium–formazan that is the color at the location of the LDH isozyme. LDH1 (H 4 ) is the most negatively charged of the isozymes and migrates closest to the anode. M 4 (LDH5) is the most positively charged isoform and migrates most closely to the cathode. The intensity of the color formed is proportional to amount of enzyme present in the spot. It is seen that LDH2 is the predominant isozyme in normal serum. When LDH1 is in a concentration higher than LDH2 in serum, myocardial infarction is suspected confirming clinical diagnosis. More recently, troponin may be measured in serum as an indicator of myocardial infarction. Elevation of LDH5 suggests the possibility of liver damage . LDH1 (H 4 ) derives from cells of the heart, LDH2 (H 3 M 1 ) derives from red blood cells and the reticuloendothelial system , LDH3 (H 2 M 2 ) derives from lungs and kidneys, LDH4 (H 1 M 3 ) derives from kidneys, and LDH5 (M 4 ) derives from liver and striated muscle.

Creatine kinase ( CPK ) is another enzyme of interest in clinical enzymology. It catalyzes the phosphorylation of creatine , the reaction of which is shown in Fig. 5.3 .

Creatine phosphate is a major energy source in muscle; hence, creatine kinase in blood can reflect degradative changes in muscle tissue. This enzyme is either a dimer being a homodimer or a mixture of two different subunits: muscle type (M) and brain type (B). Three isozymes exist: MM (CPK3), prevalent in skeletal and heart muscles; BB (CPK1) prevalent in brain, gastrointestinal tract, and genitourinary tract; and MB (CPK2), prevalent in heart muscle. In myocardial infarction, there is an increase of LDH1 over LDH2 in serum, and there are also elevations of MM and MB isozymes of creatine kinase. In skeletal muscular diseases and muscular dystrophy, the MM isozyme is elevated. Fig. 5.4 shows electrophoretic patterns of both LDH and CPK where the spots of CPK are aligned with the array of serum LDH isozymes. In line 1, for example, LDH1 is higher than LDH2, suggesting myocardial infarction. In confirmation, CPK isozymes MB and MM are elevated confirming results with LDH. CPK and CK are two abbreviations for the same enzyme (creatine phosphokinase or creatine kinase) .

Figure 5.4, Electrophoretograms of eight different measurements of LDH and CPK isozymes in serum. When LDH1 is low relative to LDH2, as in line 8, the MB and MM isozymes of CPK are also low. Compare these data to those in line 1.

In myocardial infarction, enzyme activities in serum are followed as a function of time in days after the event. Typical results for activities of LDH1, creatine kinase (MB), and aspartate aminotransferase are shown in Fig. 5.5 , and times of onset, peak of each enzymatic activity, and duration are summarized in the table of Fig. 5.5 .

Figure 5.5, Patterns of serum enzyme activities with time after myocardial infarction. Elevations in enzyme activities are observable as much as 48 h after the attack, and some activities persist for days or even weeks. AST , Aspartate aminotransferase; CK-MB , subunit of creatine kinase; heart-specific LD , LDH1, or H 4 isozyme of lactate dehydrogenase.

Blood draws are made from 18 to 30 hours after the heart attack and 12 and 48 hours beyond that. Many diseases can be confirmed or diagnosed by measurement of enzyme activities in serum, including hepatitis, jaundice, cirrhosis, muscular dystrophies, and some cancers.

Enzymes also have been used or proposed for the treatment of certain conditions. Some of these are summarized in Table 5.2 .

Table 5.2

Enzyme Preparations Used in the Treatment of Some Diseases.

Enzyme (Preparation)	Disease
Pancreatic digestive enzymes (sometimes as pancreatin or pancrelipase)	Pancreatic insufficiency
	Cystic fibrosis
(β-Glucocerebrosidase (analog) also called Cerezyme]	Gaucher disease (long-term treatment)
Hyaluronidase (human recombinant) and N -acetyl-galactosamine-4-sulfatase	Mucopolysaccharidosis VI
Myozyme (α-glucosidase)	Pompe disease (α-glucosidase deficiency)
Lactase (lactase or β-galactosidase)	Lactose intolerance
“Beano” (fungal α-galactosidase)	Prevents gas and bloating after consumption of legumes
Enzyme mixture (current research)	Celiac disease (inability to digest gluten)

General Aspects of Catalysis

Virtually, all enzymes in the body are proteins, except for ribozymes. Early life forms were probably based on nucleic acids, rather than proteins and ribozyme must be an ancient vestige and must have been among the first molecular machines. Since the discovery of an RNA that could catalyze an enzymatic reaction is recent, there is much activity into discovering other ribozymes, besides peptidyl transferase 23S rRNA (see Chapter 11: Protein Biosynthesis). There are at least 12 ribozymes known that catalyze different functions, and it is possible to design ribozymes that can cleave any RNA molecule at a specific site. However, the occurrence of ribozymes is rare. The rest of this chapter will concern enzyme proteins that are abundant in the mammalian cell.

Enzymes make possible reactions to occur under bodily conditions that otherwise would require conditions that the body could not tolerate (excessive temperature, pressure, pH, etc.). A catalyst is generally regarded as a substance, in small amount compared to the reactants, which modifies and increases the rate of a reaction without itself being consumed. This is generally true for enzymes in that they allow a reaction to occur under bodily conditions, and although they form complexes with substrates and products, they emerge from the reaction in free form just as they started out. The simplest reaction involving an enzyme (E) would be for it to catalyze the conversion of S, the substrate, being converted to P, the product of the reaction:

E + S ⇌ ES ⇌ E + P

$E + S ⇌ ES ⇌ E + P$

Note that many enzymatic reactions are reversible, as indicated by the reversible arrows, so that in the reverse reaction, the enzyme can combine with the product, P to form an enzyme–product complex (EP). In some enzymatic reactions a relatively large amount of energy is required to initiate the reaction so that the reverse reaction can be quite small compared to the forward reaction. In terms of the first reaction above, the reverse reaction would be

E + P ⇌ EP ⇌ ES ⇌ E + S

$E + P ⇌ EP ⇌ ES ⇌ E + S$

As will be seen later on, a hydrolytic reaction, where a group is split off from a molecule by the addition of water, might be more difficult to reverse than a transfer reaction where one group from one molecule is transferred to another molecule, requiring a smaller amount of energy.

So, a certain amount of energy must be invested to allow the reaction to proceed on its own in the presence of the enzyme. This is called the energy of activation ( Fig. 5.6 ).

Figure 5.6, A typical energy of activation diagram. In this example the phosphorylation of glucose is shown: glucose+ATP⇌glucose−6−phosphate+ADP glucose+ATP⇌glucose−6−phosphate+ADP . The phosphorylation of glucose by hexokinase or by glucokinase is the first reaction of glycolysis. Note that the free energy (Δ G 0 =Gibbs free energy) is the same, proceeding from the level of the substrate to the level of the product whether the enzyme is present or not. The superscript 0 is for standard reaction conditions, and these can vary dramatically from conditions inside the cell. The difference is that less energy is required to make the reaction proceed in the presence of the enzyme. EA , energy of activation.

To understand enzymatic reactions and inhibitors of enzymatic reactions, one must resort to a mathematical description of the progress of the reaction. While specifying the conditions of the reaction (pH and buffer, temperature, salt concentration, etc.), the simplest reaction is that of a single substrate to produce a single product (S going to P). When measurements are taken as a function of time, the rate or velocity (ordinate or y -axis) can be plotted as a function of substrate concentration ([S], abscissa or x -axis) as shown in Fig. 5.7 .

Figure 5.7, Velocity ( v ) of an enzymatic reaction ( y -axis or ordinate) as a function of substrate concentration ([S]) ( x -axis or abscissa). Velocity can be quantified as the rate of appearance of the product, P, for which there is usually a direct or indirect measurement. When the various time points are plotted, a first-order curve is obtained as shown in the figure. In this figure the actual time points are not shown. The straight-line portion of the curve (at lower substrate concentrations) represents a zero-order reaction where the rate of the reaction is proportional to the substrate concentration. The curve reaches saturation (levels off) at the maximal velocity ( V max ). At the half-maximal velocity the value of the molar substrate concentration ([S]) is equal to the Michaelis–Menten constant for the given reaction.

The changes in the components of the first-order reaction are shown in Fig. 5.8 .

Figure 5.8, Rates of change of the concentrations of substrate (S), product (P), enzyme (E), and enzyme–substrate complex (ES). The substrate concentration, [S], is in great excess over the amount of enzyme, [E]. The concentration of the enzyme–substrate complex, [ES], is low at the start of the reaction, and it is assumed that it remains constant until the end of the reaction when the concentration of substrate is small. CONC , Concentration.

The data of the first-order reaction curve ( Fig. 5.7 ) can be represented in the form of a straight line when the reciprocal of the initial velocity (1/ v i ) is plotted on the ordinate ( y -axis) as a function of the reciprocal of the substrate concentration (1/[S]) plotted on the x -axis (abscissa). This representation is known as the Lineweaver–Burk plot , and it is shown in Fig. 5.9 .

Figure 5.9, Lineweaver–Burk plot of a first-order enzymatic reaction. 1/ v i is plotted on the ordinate as a function of the reciprocal substrate concentration, 1/[S], plotted on the abscissa. In this case the value of the Michaelis–Menten constant , K m , is found when the line extrapolates to the second quadrant on the x -axis where the intersection is −1/ K m . The K m is equal to the substrate concentration that promotes 50% of maximal velocity of the reaction. The value of the maximal velocity is determined at the point of intersection on the ordinate that is equal to 1/ V max . Knowing the value of V max from this point, K m also can be calculated from the slope that is equal to K m / V max .

Lineweaver–Burk Equation

The Lineweaver–Burk equation is derived from the Michaelis–Menten equation (elaborated next) and has the form:

1 / v = (K_{m} (1 / V_{\max})) ([S]) + 1 / V_{\max}

$1 / v = (K_{m} (1 / V_{\max})) ([S]) + 1 / V_{\max}$

1 / v = (K_{m} / V_{\max}) (1 / [S]) + 1 / V_{\max}

$1 / v = (K_{m} / V_{\max}) (1 / [S]) + 1 / V_{\max}$

that is written in the form of a straight-line equation :

y = mx + b

$y = mx + b$

y is the ordinate value, m is the slope of the straight line, x is the abscissa value, and b is the value at the intersection on the y -axis (when x =0; see Fig. 5.9 ). The intersection on the x -axis is −1/ K _m . The straight-line representation facilitates the measurements of K _m , and V _max compared to the direct plot ( Fig. 5.7 ). Interestingly, the K _m value approximates the molar substrate concentration present in the cell.

Michaelis–Menten Equation

In the simple enzymatic reaction,

E + S \overset{k_{1}}{\underset{k_{- 1}}{⇌}} ES \overset{k_{2}}{\to} P + E

$E + S \overset{k_{1}}{\underset{k_{- 1}}{⇌}} ES \overset{k_{2}}{\to} P + E$

The net effect of the reaction is the conversion of S to P. The term, E, being on both sides of the equation, drops out, reflecting its catalytic action. The enzyme increases the rate of the reaction but is, itself, not altered.

The initial velocity of the reaction, v ₀ , is the rate of appearance of product, P, as a function of time:

v_{0} = {(\frac{d [P]}{dt})}_{0}

$v_{0} = {(\frac{d [P]}{dt})}_{0}$

and v ₀ is also proportional to the rate of formation of the enzyme–substrate (ES) complex, ES, and its breakdown to form product:

v_{0} = d [P] / dt = k_{2} [ES] = k_{2} {[E]}_{T} [S] / K_{m} + [S]

$v_{0} = d [P] / dt = k_{2} [ES] = k_{2} {[E]}_{T} [S] / K_{m} + [S]$

[E] _T is the total amount of enzyme placed into the reaction, and K _m is the Michaelis constant (as measured in Fig. 5.9 ). It is the ratio of the rates of the reactions leading to product formation to the rate of the reverse reaction:

K_{m} = k_{1} + k_{2} / k_{- 1}

$K_{m} = k_{1} + k_{2} / k_{- 1}$

When the substrate concentration, [S], is increased significantly so that the rate of reaction is no longer limited by [S], v ₀ approaches maximal velocity, V _max , so that, in the equation,

v_{0} = k_{2} {[E]}_{T} [S] / K_{m} + [S]

$v_{0} = k_{2} {[E]}_{T} [S] / K_{m} + [S]$

all terms in S drop out, including K _m (which is a substrate concentration), then

V_{\max} = k_{2} {[E]}_{T} = constant

$V_{\max} = k_{2} {[E]}_{T} = constant$

As V _max = k ₂ [E], V _max can substitute for k ₂ [E] as follows:In

v_{0} = k_{2} {[E]}_{T} [S] / K_{m} = [S]

$v_{0} = k_{2} {[E]}_{T} [S] / K_{m} = [S]$

substituting V _max for k ₂ [E], the Michaelis–Menten equation becomes,

v_{0} = V_{\max} [S] / K_{m} + [S]

$v_{0} = V_{\max} [S] / K_{m} + [S]$

This equation describes the plot in Fig. 5.9 .

The Lineweaver–Burk equation, describing the straight-line plot shown in Fig. 5.9 , is derived from the Michaelis–Menten equation by taking the reciprocals of both sides of the equation. Thus

v_{0} = V_{\max} [S] / K_{m} + [S]

$v_{0} = V_{\max} [S] / K_{m} + [S]$

becomes

\frac{1}{v_{0}} = K_{m} / V_{\max} [S] + [S] / V_{\max} [S]

$\frac{1}{v_{0}} = K_{m} / V_{\max} [S] + [S] / V_{\max} [S]$

and

\frac{1}{v_{0}} = K_{m} / V_{\max} 1 / [S] + 1 / V_{\max}

$\frac{1}{v_{0}} = K_{m} / V_{\max} 1 / [S] + 1 / V_{\max}$

is in the form of a straight-line equation:

y = mx + b

$y = mx + b$

making the determination of K _m and V _max direct.

The number of ES complexes converted to product per enzyme molecule per unit time is the turnover number of the enzyme ( k _cat ). The rate of formation of E+P essentially determines the conversion of the ES complex to product, this rate, k ₂ , approximates k _cat :

k_{cat} = k_{2} = \frac{k_{2} {(E)}_{T}}{{(E)}_{T}} = \frac{V_{\max}}{{(E)}_{T}} = turnover number

$k_{cat} = k_{2} = \frac{k_{2} {(E)}_{T}}{{(E)}_{T}} = \frac{V_{\max}}{{(E)}_{T}} = turnover number$

The turnover number is expressed as s ^{− 1} , or reciprocal seconds (1/s). The turnover number informs on the rate of conversion of substrate to product that can be useful information for a given enzyme.

Inhibition of Enzymatic Activity

Within a cell in the body, there may be more than 3000 different enzymes. The rates of many of these enzymes are regulated by other molecules in the cell, and these other molecules participate in the homeostatic milieux. Sometimes, an initial or early enzyme in a metabolic pathway is the rate-limiting step for the function of the entire pathway, and the regulation of this enzyme is of special importance. Sometimes, the activity of an enzyme like this is under allosteric control (to be discussed subsequently). Of great interest is the use of medicines/drugs, which function by the inhibition of specific enzymatic activity. Examples of specific drugs will be mentioned later.

There are two types of inhibition of enzymes. One is reversible in which the inhibitor binds to the enzyme noncovalently. In irreversible inhibition the inhibitor binds covalently to the enzyme. A reversible inhibitor can bind to the active site of the enzyme where the substrate binds. Consequently, this inhibition is reversible by increasing the amount of the substrate to compete with the inhibitor. Covalent binding of the inhibitor to the enzyme, at any site, is noncompetitive. Also, noncompetitive inhibition can be obtained when a noncovalent inhibitor binds so strongly that the reaction is reversible only to a limited extent.

In addition to competitive and noncompetitive inhibition, a third type that is less common is uncompetitive inhibition .

Competitive Inhibition

This type of inhibition is the most important for the use of medicines. A type of drug is desired that can titrate (dosage) the activity of an enzyme competitively while still having the advantage of being able to withdraw the drug and allow the enzymatic activity to recover. This would be in contrast to a noncompetitive drug inhibitor that would tie up the enzyme in a dead-end complex , and withdrawal of the drug would not allow the enzyme to resume its normal activity. Obviously, a drug that is a competitive inhibitor would be the medicine of choice in most cases.

The characteristic of a competitive inhibitor is that it binds in the same site as the substrate and, therefore, competes with the substrate for the enzyme’s binding site or substrate pocket. This is visualized in Fig. 5.10 .

Figure 5.10, A model showing that a competitive inhibitor binds to the substrate-binding site.

A competitive inhibitor resembles the structure of the substrate. Most drugs are competitive inhibitors that bind to the active site of an enzyme more strongly than the substrate. The inhibition by this type of inhibitor is reversible by increasing the amount of available substrate. When the concentration of the inhibitor is increased, the rate of the reaction will decrease accordingly because a greater number of the active sites of the enzyme will be occupied by the inhibitor rather than by the substrate. A plot of the time course of an enzymatic reaction in the presence or absence of a competitive inhibitor is shown in Fig. 5.11 .

Figure 5.11, Velocity of the enzymatic reaction is plotted on the y -axis (ordinate) versus the substrate concentration on the x -axis (abscissa). The values of K m and v are altered but V max is unaffected (theoretically, if the reaction were to be carried out long enough the rate of the inhibited reaction would reach the same rate as the uninhibited reaction; the inhibited reaction would take much longer to reach the maximal velocity). Or, putting it another way, the same V max can be achieved with the competitive inhibitor present but a higher concentration of substrate would be required. The K m observed in the presence of the inhibitor is modified by the quantity, 1+[I]/ K i , where [I] is the molar concentration of inhibitor, and K i is the inhibition constant of the inhibitor.

If Fig. 5.11 showed a continuing higher amount of substrate on the x -axis, the curve for the presence of the competitive inhibitor would reach the same value as the control ( V _max ) with no inhibitor emphasizing that increased substrate can overcome (replace) the competitive inhibitor. The inhibition constant, K _i , is defined as the dissociation constant in the reversible reaction between enzyme and inhibitor :

E + I ⇌ EI

$E + I ⇌ EI$

Therefore

K_{i} = [E] [I] / [EI]

$K_{i} = [E] [I] / [EI]$

The double reciprocal plot of the reaction shown in Fig. 5.11 is shown in Fig. 5.12 .

Figure 5.12, Double reciprocal (Lineweaver–Burk) plot of a reaction similar to that shown in Fig. 5.11 . The line with the inhibitor is raised above the normal reaction without inhibitor since this is a reciprocal plot. The straight-line equation is modified by the quantity 1+[I]/ K i in the presence of the competitive inhibitor, applying to the slope ( K m / V max ) and the value of the x -axis (1/[S]) intercept as shown by the equation in the figure.

As shown in Fig. 5.12 , in the presence of a competitive inhibitor , the slope and the value of the x -axis intercept are increased by 1+[I]/ K _i :

1 / v = K_{m} / V_{\max} (1 / [S]) + 1 / V_{\max}

$1 / v = K_{m} / V_{\max} (1 / [S]) + 1 / V_{\max}$

becomes

1 / v = K_{m} / V_{\max} (1 + [I] / K_{i}) (1 / [S]) (1 + [I] / K_{i}) + 1 / V_{\max}

$1 / v = K_{m} / V_{\max} (1 + [I] / K_{i}) (1 / [S]) (1 + [I] / K_{i}) + 1 / V_{\max}$

1 / v = K_{m} / V_{\max} [S] (1 + [I] / K_{i}) + 1 / V_{\max}

$1 / v = K_{m} / V_{\max} [S] (1 + [I] / K_{i}) + 1 / V_{\max}$

but the value of V _max is unchanged.

Noncompetitive Inhibition

In the case of a noncompetitive inhibitor the binding of the inhibitor is often to a site distant from the substrate-binding site (the active site). Here, all of the components of the equation are modified by the value, 1+[I]/ K _i :

1 / v = K_{m} / V_{\max} [S] (1 + [I] / K_{i}) + 1 / V_{\max} (1 + [I] / K_{i})

$1 / v = K_{m} / V_{\max} [S] (1 + [I] / K_{i}) + 1 / V_{\max} (1 + [I] / K_{i})$

A comparison of the initial velocity, v , as a function of substrate concentration ([S]) between the two types of inhibition is shown in Fig. 5.13 .

Figure 5.13, Initial velocity, v i , is plotted on the ordinate ( y -axis) as a function of the molar concentration of substrate ([S]) on the x -axis (abscissa). As indicated previously, if the reaction with the competitive inhibitor progresses long enough, it will eventually reach the value of the V max . This is not the case in the presence of a noncompetitive inhibitor where some of the enzyme is actually removed from the reaction by forming the dead-end complex, EI, and V max is lower.

The inhibition by a noncompetitive inhibitor will not be reversed by increasing the concentration of substrate because the inhibitor binding site is distant from the substrate-binding site, and the inhibitor’s structure may not resemble that of the substrate; thus the reaction will remove enzyme from the productive pathway:

This situation is pictured in Fig. 5.14 .

Figure 5.14, A model of noncompetitive inhibition indicating that the substrate and the noncompetitive inhibitor bind at different sites on the enzyme.

When the noncompetitive inhibitor forms a covalent bond with the inhibitor site on the enzyme, the enzyme is essentially irreversibly removed from the reaction (forming a dead-end complex). However, even if the inhibitor binds to the enzyme covalently, there may be a trickle of reversibility (principle of microscopic reversibility). In general, a reversible inhibitor binds to its site noncovalently generating a forward as well as a reverse reaction. If the reversible inhibitor binds to the active site (where the substrate binds), increasing the amount of substrate will displace the reversible (competitive) inhibitor. A covalent inhibitor has a tiny or nonmeasurable reverse reaction so that it essentially removes the enzyme from the reaction by forming a dead-end complex. The double reciprocal plot of noncompetitive inhibition is shown in Fig. 5.15 .

Figure 5.15, Double reciprocal plot of noncompetitive inhibition. The upper line represents the system in the presence of the inhibitor (the line is increased because this is a reciprocal plot). The slope and 1/ V max values are modified by the term, 1+[I]/ K i as shown by the equation in the figure. The x -axis intercept (in the second quadrant) is unchanged by the presence of the inhibitor. This is not true for competitive inhibition ( Fig. 5.12 ) where the value of the x -axis intercept is increased in the presence of the inhibitor (less negative, second quadrant). The y -axis intercept in the presence of the inhibitor is moved upward, whereas, in competitive inhibition, it is unchanged.

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Diagnostic Enzymology

Enzymes With Multiple Subunits: Tissue-Specific Isozymes

General Aspects of Catalysis

Lineweaver–Burk Equation

Michaelis–Menten Equation

Inhibition of Enzymatic Activity

Competitive Inhibition

Noncompetitive Inhibition

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