Enzymes PDF - Clinical Significance and Classification

CHAPTER 8 © Yurchanka Siarhei/Shutterstock. Enzymes Donna J. Spannaus-Martin CHAPTER OUTLINE General Properties and Definitions Aspartate Aminotransferase...

CHAPTER 8 © Yurchanka Siarhei/Shutterstock. Enzymes Donna J. Spannaus-Martin CHAPTER OUTLINE General Properties and Definitions Aspartate Aminotransferase Alanine Aminotransferase Enzyme Classification and Nomenclature Alkaline Phosphatase Enzyme Kinetics Acid Phosphatase Enzyme Catalysis -Glutamyltransferase Factors That Influence Enzymatic Reactions 5-Nucleotidase Measurement of Enzyme Activity Amylase Calculation of Enzyme Activity Lipase Measurement of Enzyme Mass Glucose-6-Phosphate Dehydrogenase Enzymes as Reagents Macroenzymes Enzymes of Clinical Significance Drug-Metabolizing Enzymes Creatine Kinase References Lactate Dehydrogenase KEY TERMS Activation energy Holoenzyme Ligases Activators Hydrolase Lyases Apoenzyme International unit (IU) Michaelis-Menten constant Coenzyme Isoenzyme Oxidoreductase Cofactor Isoform Transferases Enzyme–substrate (ES) complex Isomerases Zero-order kinetics First-order kinetics Kinetic assays Zymogen CHAPTER OBJECTIVES Upon completion of this chapter, the clinical laboratorian should be able to do the following: Define the term enzyme, including physical Explain why the measurement of plasma enzyme composition and structure. concentrations is clinically useful. Classify enzymes according to the International Union Differentiate which enzymes are useful in the diagnosis of Biochemistry. of various disorders, including cardiac, hepatic, bone, List the different factors affecting the rate of an and muscle malignancies, and acute pancreatitis. enzymatic reaction. Discuss the tissue sources, diagnostic significance, Diagram enzyme kinetics, including zero-order and clinical assays, and the sources of error for first-order kinetics. the following enzymes: creatine kinase, lactate 225 226 Chapter 8 Enzymes dehydrogenase, aspartate aminotransferase, alanine Evaluate patient plasma enzyme concentrations in aminotransferase, alkaline phosphatase, acid relation to disease states. phosphatase, γ-glutamyltransferase, 5'-nucleotidase, State the clinical importance for detecting amylase, lipase, and glucose-6-phosphate macroenzymes. dehydrogenase. Specify the role of enzymes in drug metabolism. Enzymes are specific proteins that catalyze biochem- and turns (secondary structure), and folding into a ical reactions without altering the equilibrium point three-dimensional structure (tertiary structure) that of the reaction or being consumed or changed in results in structural features such as binding cavities. composition. The other substances in the reaction, If an enzyme contains more than one polypeptide substrates, are converted to products. The catalyzed unit, each polypeptide is called a subunit and the reactions are frequently specific and essential to phys- quaternary structure refers to the binding and inter- iologic functions, such as the hydration of carbon actions between the subunits. Each enzyme contains dioxide, muscle contraction, nutrient degradation, an active site, often a water-free cavity, where the sub- and energy use. Found in all body tissues, enzymes strate interacts with particular amino acid residues. frequently appear in the serum following cellular An allosteric site, a cavity other than the active site, injury, or sometimes from degraded cells, in smaller binds regulator molecules and, thereby, may be influ- amounts. Certain enzymes, such as those that facili- entially significant to the basic enzyme structure. tate coagulation, are specific to plasma and, therefore, Even though a particular enzyme maintains the are present in significant concentrations in plasma. same catalytic function throughout the body, differ- Plasma or serum enzyme levels are often useful in the ent forms of that enzyme may exist in various types diagnosis of particular diseases or physiologic abnor- of tissue within the same individual. The different malities. This chapter discusses the general proper- forms may differ in select physical properties, such ties and principles of enzymes, aspects relating to the as electrophoretic mobility, solubility, or resistance clinical diagnostic significance of specific physiologic to inactivation. The term isoenzyme is generally enzymes, and assay methods for those enzymes. used when discussing such forms of the enzymes, although the International Union of Biochemistry (IUB) suggests restricting this term to multiple forms General Properties of similar genetic origin. An isoform results when and Definitions an enzyme is subject to posttranslational modifica- tions with a functional group added to an amino acid. Enzymes catalyze many specific reactions. These reac- Isoenzymes and isoforms contribute to heterogene- tions are facilitated by the enzyme structure and sev- ity in properties and function of enzymes because eral other factors. As a protein, each enzyme contains these measured properties are influenced by changes a specific amino acid sequence (primary ­structure), in amino acid chemistry and the resulting changes with the resultant polypeptide chains adopting bends in structural ­features. CASE STUDY 8.1, PART 1 Let’s meet Carl, a 57-year-old moderately overweight White male. Carl visits his family phy- sician with a symptom of “indigestion” of 5 days’ duration. He has also had bouts of sweating, malaise, and headache. His blood pressure is 140/105 mm Hg; his family history includes a father who died at age 62 of acute myocardial infarction (AMI) secondary to diabetes mellitus. © Tony Wear/Shutterstock.  Enzyme Classification and Nomenclature 227 In addition to the basic enzyme structure, a non- many systematic names are lengthy, a more usable, protein molecule, called a cofactor, may be neces- trivial, recommended name is also assigned by the IUB sary for enzyme activity. Inorganic cofactors, such system.1 as metal ions, are called activators. A coenzyme In addition to naming enzymes, the IUB system is an organic cofactor, such as nicotinamide adenine identifies each enzyme by an EC numerical code con- dinucleotide (NADH). When bound tightly to the taining four digits separated by decimal points. The enzyme, the coenzyme is called a prosthetic group. first digit places the enzyme in one of the following The protein portion (apoenzyme), binding its six classes: respective coenzyme, forms a complete and active 1. Oxidoreductases catalyze an oxidation–reduc- system, a h ­ oloenzyme. tion reaction between two substrates. Some enzymes, mostly digestive enzymes, are 2. Transferases catalyze the transfer of a group originally secreted from the organ of production in other than hydrogen from one substrate to a structurally inactive form, called a proenzyme or another. zymogen. The zymogen form is thus prevented from 3. Hydrolases catalyze hydrolysis of various bonds. acting until needed. The zymogen can be activated by 4. Lyases catalyze removal of groups from sub- other enzymes that alter the structure of the proen- strates without hydrolysis; the product contains zyme, typically with the removal of some sequence double bonds. that masks the active form. For example, this mech- 5. Isomerases catalyze the interconversion of geo- anism prevents digestive enzymes from digesting the metric, optical, or positional isomers. tissue where they are synthesized. 6. Ligases catalyze the joining of two substrate molecules, coupled with breaking of the pyro- phosphate bond in adenosine triphosphate (ATP) Enzyme Classification or a similar compound. and Nomenclature The second and third digits of the EC code num- ber represent the subclass and sub-subclass of the To standardize enzyme nomenclature, the Enzyme enzyme, respectively, divisions that are made accord- Commission (EC) of the IUB adopted a classifica- ing to criteria specific to the enzymes in the class. tion system in 1961, with the standards revised in The final number is the serial number specific to 1972 and 1978. The IUB system assigns a systematic each enzyme in a sub-subclass. Table 8.1 provides name to each enzyme, defining the substrate acted the EC code numbers, as well as the systematic and on, the reaction catalyzed, and, possibly, the name recommended names, for enzymes frequently mea- of any coenzyme involved in the reaction. Because sured in the clinical laboratory. Table 8.1 Classification of Frequently Quantified Enzymes Recommended Common Standard Class Name Abbreviation Abbreviation EC Code No. Systematic Name Oxidoreductases Lactate LD LD 1.1.1.27 l-Lactate:NAD+ dehydrogenase oxidoreductase Glucose-6- G-6-PDH G-6-PD 1.1.1.49 d-Glucose-6- phosphate phosphate:NADP+ dehydrogenase 1-oxidoreductase Glutamate GLD GLD 1.4.1.3 l-Glutamate:NAD(P) dehydrogenase oxidoreductase, deaminase Transferases Aspartate amino GOT (glutamate AST 2.6.1.1 l-Aspartate:2-oxoglutarate transferase oxaloacetate aminotransferase transaminase) Alanine amino- GPT (glutamate ALT 2.6.1.2 l-Alanine:2-oxoglutarate transferase transaminase) aminotransferase (continues) 228 Chapter 8 Enzymes Table 8.1 Classification of Frequently Quantified Enzymes(continued) Recommended Common Standard Class Name Abbreviation Abbreviation EC Code No. Systematic Name Creatine kinase CPK (creatine CK 2.7.3.2 ATP:creatine phosphokinase) N-phosphotransferase γ-Glutamyl- GGTP GGT 2.3.2.2 (5-Glutamyl)peptide: amino transferase acid-5-glutamyltransferase Glutathione-S- α-GST GST 2.5.1.18 Glutathione transferase transferase Glycogen GP GP 2.4.1.1 1,4-d-Glucan: phosphorylase orthophosphate α-d- glucosyltransferase Pyruvate kinase PK PK 2.7.1.40 Pyruvate kinase Hydrolases Alkaline ALP ALP 3.1.3.1 Orthophosphoric phosphatase monoester phosphohydrolase (alkaline optimum) Acid phosphatase ACP ACP 3.1.3.2 Orthophosphoric monoester phosphohydrolase (acid optimum) α-Amylase AMY AMS 3.2.1.1 1,4-d-Glucan glucanohydrolase Cholinesterase PCHE CHE 3.1.1.8 Acetylcholine acyl- hydrolase Chymotrypsin CHY CHY 3.4.21.1 Chymotrypsin Elastase-1 E1 E1 3.4.21.36 Elastase 5′-Nucleotidase NTP 5NT 3.1.3.5 5′-Ribonucleotide phosphohydrolase Triacylglycerol LPS 3.1.1.3 Triacylglycerol lipase acylhydrolase Trypsin TRY TRY 3.4.21.4 Trypsin Lyases Aldolase ALD ALD 4.1.2.13 d-d-Fructose- 1,6-bisdiphosphate d- glyceraldehyde3- phosphate-lyase Isomerases Triosephosphate TPI TPI 5.3.1.1 Triosephosphate isomerase isomerase Ligase Glutathione GSH-S GSH-S 6.3.2.3 Glutathione synthase synthetase Data from Competence Assurance, ASMT. Enzymology, An Educational Program. Bethesda, MD: RMI Corporation; 1980. Table 8.1 also lists common and standard abbre- accepted names for the enzymes, were used until viations for commonly analyzed enzymes. Without the standard abbreviations listed in the table were IUB recommendation, capital letters have been used ­developed.2,3 These standard abbreviations are used as a convenience to identify enzymes. The common in the United States and are used later in this chapter abbreviations, sometimes developed from previously to indicate specific enzymes.  Enzyme Kinetics 229 Enzyme Kinetics One way to provide more energy for a reaction is to increase the temperature, which will increase inter- Enzyme Catalysis molecular collisions; however, this does not normally Some chemical reactions will occur at a slow rate occur physiologically. Enzymes catalyze physiologic if there is not enough kinetic energy to drive the reactions by lowering the activation energy level that reaction to the formation of products (uncatalyzed the reactants (substrates) require to form the transi- ­reaction). Other chemical reactions can occur spon- tion state, allowing the reaction to occur (Figure 8.1). taneously if the free energy or available kinetic The reaction may then more readily achieve a state energy is higher for the reactants than for the prod- of equilibrium; the extent to which the reaction pro- ucts. An obstacle that slows the reaction is often ceeds to product depends on the number of substrate the formation of the transition state of the reaction. molecules that pass the energy barrier. The transition state is the intermediate state formed The general relationship among enzyme, substrate, after the reactants exist, but before the product is and product may be represented as follows: formed and energy is required for the formation E + S → ES → E + P (Eq. 8.1) of the transition state and conversion to product. The reaction proceeds toward the lower energy if where E is enzyme, S is substrate, ES is enzyme–­ a sufficient number of the reactant molecules pos- substrate complex, and P is product. sess enough excess energy to break their chemical The ES complex is a physical association of a bonds and collide to form new bonds. The excess substrate to the active site of an enzyme. The struc- energy needed to induce the transition state is called tural arrangement of amino acid residues within the ­ ctivation energy of the reaction and is the energy a enzyme directs the formation of secondary structures required to raise all molecules in one (1) mole of and guides the formation of its three-dimensional a compound at a certain temperature to the transi- active site. Enzymes are induced to form a tighter tion state at the peak of the energy barrier. Reactants active structure after the binding of substrate drives a possessing enough energy to overcome the energy rearrangement, resulting in enhanced substrate bind- barrier result in product formation. ing; this is the Induced Fit model of enzyme action. Energy barrier Activation energy for uncatalyzed reaction Energy barrier Activation energy for catalyzed reaction Initial reaction stage Net Free energy free energy decrease Equilibrium Reaction Figure 8.1 Energy versus progression of reaction, indicating the energy barrier that the substrate must surpass to react with and without enzyme catalysis. The enzyme considerably reduces the free energy needed to activate the reaction. © Wolters Kluwer. 230 Chapter 8 Enzymes The transition state for the ES complex has a lower energy of activation to form the transition state than S alone, so that the reaction proceeds more effectively after the ES complex is formed. An actual reaction may involve several substrates and products. Different enzymes show specificity to substrates in different extents or respects. Certain enzymes exhibit absolute specificity, meaning that the enzyme combines with only one substrate and catalyzes only the one corresponding reaction. Other enzymes are group specific because they combine with all substrates containing a particular functional group, such as a phosphate ester. Still other enzymes are specific to chemical bonds and thereby exhibit bond specificity. Km Stereoisomeric specificity refers to enzymes that predominantly combine with only one optical iso- Figure 8.2 Michaelis-Menten curve of velocity versus substrate concentration for enzymatic reaction. Km is the mer of a certain compound. For example, an enzyme substrate concentration at which the reaction velocity is may bind more than one molecule of substrate, and half of the maximum level. this may occur in a cooperative fashion. Binding of © Wolters Kluwer. one substrate molecule, therefore, may facilitate binding of additional substrate molecules. Likewise, velocity has reached its maximum. When product some enzymes may act on more than one substrate is formed, the resultant free enzyme immediately and have preferred binding order for the substrates. becomes available to combine with available free In some examples, binding may occur via an ordered substrate. At this point the reaction is in zero-­order binding sequence in which one substrate must bind kinetics, and the reaction rate depends only on first before the second substrate can bind for the reac- enzyme concentration. tion to proceed. Other enzymes show a random order The Michaelis-Menten hypothesis describes the of substrate binding in which either of two substrates relationship between reaction velocity and substrate can bind first, followed by the other substrate driving concentration, which is represented mathematically the reaction to form the product. as follows: Vmax [S] Factors That Influence V= K m + [S] (Eq. 8.2) Enzymatic Reactions where V is measured velocity of reaction, Vmax is Substrate Concentration maximum velocity, [S] is substrate concentration, The rate at which an enzymatic reaction proceeds and Km is Michaelis-Menten constant of enzyme for and whether the forward or reverse reaction occurs a specific substrate. can be influenced by several reaction conditions. The Michaelis-Menten constant (Km), derived One major influence on enzymatic reactions is sub- from the theory of Michaelis and Menten, is a constant strate concentration. In 1913, Leonor Michaelis and for a specific enzyme and substrate under defined Maud Menten hypothesized the role of substrate con- reaction conditions and can provide an expression of centration in formation of the enzyme–­substrate the relationship between the velocity of an enzymatic (ES) ­complex. According to their hypothesis, rep- reaction and substrate concentration. The assump- resented in F­igure 8.2, the substrate readily binds tions are made that an equilibrium among E, S, ES, to free enzyme at a low substrate concentration. and P is established rapidly and that the reverse reac- The reaction rate steadily increases when more sub- tion, E + P → ES, r is negligible. The rate-limiting strate is added as substrate increasingly associates step is the formation of product and enzyme from the with enzyme. During this process, the reaction is ES complex (ES → E + P). When reached, maximum following first-order kinetics because the rate velocity is fixed, and the reaction rate is a function of the reaction is directly proportional to substrate of only the enzyme concentration. A specific variable, concentration. If the substrate concentration is high the Michaelis Constant, or Km, is characteristic of each enough to saturate all available enzyme, the reaction enzyme-substrate pair under the reaction conditions  Enzyme Kinetics 231 being used and is determined by a series of rate con- K of m. The intercept on the y-axis is the value of stants for the reaction. As designated in Figure 8.2, V Km is specifically the substrate concentration at which 1 max for the reaction and the intercept on the x-axis the enzyme yields half the possible maximum velocity. Vmax –1 Therefore, Km reflects the amount of substrate needed provides a value of for the reaction. Km for a particular enzymatic reaction. Vmax [S] Enzyme Concentration V= Because enzymes catalyze physiologic reactions, the K m + [S] enzyme concentration affects the rate of the cata- Theoretically, Vmax and then Km could be deter- lyzed reaction. As long as the substrate concentra- mined from the plot in Figure 8.2. However, Vmax is tion exceeds the enzyme concentration, the velocity difficult to determine from the hyperbolic plot and of the reaction is proportional to the enzyme con- often not actually achieved in enzymatic reactions. A centration. The higher the enzyme concentration, more accurate and convenient determination of Vmax the faster the reaction will proceed because more and Km is made through mathematical manipulations enzyme is available to bind substrate. of the Michaelis-Menten equation to yield a linear equation. The Lineweaver-Burk plot, a double recip- pH rocal plot of the Michaelis-Menten equation, yields a Enzymes are proteins that carry net molecular straight line (Figure 8.3) after the reciprocal is taken of charges because many of the amino acid side chains both the substrate concentration term and the velocity carry a functional group capable of carrying an term of the Michaelis-Menton reaction. The equation ionization charge. As such, changes in the pH of becomes: the solution can influence the ionization state of 1 K 1 1 the enzyme. Changes in pH may also denature an = m × + (Eq. 8.3) enzyme or influence its ionic state, resulting in struc- V Vmax [S] Vmax tural changes or a change in the charge on amino acid 1 residues in the active site, affecting enzyme function. Plotting the reciprocal of the velocity on the Hence, each enzyme operates within a specific pH V y-axis vs. the reciprocal of the substrate concentration range and maximally at a specific pH. Most physi- 1 ologic enzymatic reactions occur in the pH range of on the x-axis provides a straight line with a slope 7.0 to 8.0, but some enzymes are active in wider pH [S] ranges than others. In the laboratory, the pH for a reaction is carefully controlled at the optimal pH by means of appropriate buffer solutions. Temperature Increasing temperature usually increases the rate of V a chemical reaction by increasing the movement of molecules, the rate at which intermolecular colli- sions occur, and the energy available for the reaction enabling achieving the transition state and providing sufficient activation energy. Typically, for each 10°C increase in temperature, the rate of the reaction will approximately double. This is likewise the case with Vmax enzymatic reactions, unless the temperature is high Km enough to cause denaturation of the protein struc- ture of the enzyme. Figure 8.3 Lineweaver-Burk transformation of Each enzyme functions optimally at a particular Michaelis-Menten curve. Vmax is the reciprocal of the temperature, which is influenced by other reaction x-intercept of the straight line. Km is the negative variables, especially the total time for the reaction. reciprocal of the x-intercept of the same line. The optimal temperature is usually close to that of © Wolters Kluwer. the physiologic environment of the enzyme; ­however, 232 Chapter 8 Enzymes some denaturation may occur at the human physio- When quantifying an enzyme that requires a particu- logic temperature of 37°C. The rate of denaturation lar cofactor, that cofactor should always be provided increases as the temperature increases and usually is in excess so that the extent of the reaction does not significant at 40°C to 50°C. Enzymes show various depend on the concentration of the cofactor. dependencies on temperature related to the thermo- dynamic properties involved in their secondary and Inhibitors tertiary structures. Because low temperatures render enzymes revers- The primary basis for an enzymatic reaction is the ini- ibly inactive, many serum or plasma specimens for tial association with its substrate. Enzymatic reactions enzyme measurement are refrigerated or frozen to may not progress normally if a particular substance, prevent activity loss until analysis. Storage proce- an inhibitor, interferes with the reaction. Enzyme– dures may vary from enzyme to enzyme because of inhibitor interactions are reversible, reaching an equi- individual stability characteristics. Repeated freezing librium depending on the concentration of the inhib- and thawing, however, tends to denature protein and itor. A compound that shares some structural feature should be avoided. found in the substrate will typically physically bind to Because of their temperature sensitivity, enzymes the same form of the enzyme that the substrate binds, should be analyzed under strictly controlled tempera- often in the active site of an enzyme, and compete ture conditions. Incubation temperatures should be with the substrate for a place in the active site. Such accurate within ±0.1°C. Laboratories usually attempt inhibitors are called competitive inhibitors, usually to establish an analysis temperature for routine enzyme binding to the free form of the enzyme, E. With a sub- measurement of 25°C, 30°C, or 37°C. Attempts to strate concentration significantly higher than the con- establish a universal temperature for enzyme analy- centration of the competitive inhibitor, the inhibition sis have been unsuccessful, and therefore, reference is reversible because the substrate is more likely than ranges for enzyme levels may vary significantly among the inhibitor to bind in the active site and the enzyme laboratories. In the United States, however, 37°C is structure has not been destroyed by the inhibitor. most commonly used. Enzymes may also be sensitive to the presence of other compounds that are called noncompetitive inhib- itors. Most commonly, these inhibitors associate with Cofactors enzymes at a place other than the active site showing Simple cofactors are nonprotein entities that often allosteric inhibition. Noncompetitive inhibitors may bind to particular enzymes and provide a necessary allow substrate binding but will inhibit the forma- function before a reaction occurs. Common activa- tion of product. In this situation, the inhibitor may tors (inorganic cofactors) are metallic (Ca2+, Fe2+, bind to both the enzyme (E) and enzyme–substrate Mg2+, Mn2+, Zn2+, and K+). The activator may be complex (ES) with the same affinity. A related type essential for the reaction or may only enhance the of inhibitor may be identified as a mixed inhibitor reaction rate in proportion with concentration. Acti- with the capacity to bind both the E and ES forms vators function via various mechanisms, including of the enzyme, but with different affinities for the alternating the spatial configuration of the enzyme two forms. A third pattern of inhibition, uncompeti- for proper substrate binding, linking substrate to the tive inhibition, may be observed in which the inhibitor enzyme or coenzyme, or undergoing oxidation or binds only to the ES complex; increasing substrate reduction. concentration results in more ES complexes to which Organic cofactors, called coenzymes, include the inhibitor binds and, thereby, increases the inhibi- compounds such as nucleotide phosphates and vita- tion. The enzyme–substrate–inhibitor complex (ESI) mins, and often serve a more specialize function for does not yield product. In these situations, binding enzymes. When bound tightly to the enzyme, coen- of an inhibitor to the free E or ES complex is defined zymes are called prosthetic groups. Coenzymes also by an equilibrium constant for the formation of an EI often serve as second substrates for enzymatic reac- or ESI complex. tions. For example, as a cofactor, nicotinamide adenine The various types of inhibitors may be distin- dinucleotide as NAD+ may be reduced to NADH in a guished by the changes identified in the double-­ reaction in which the primary substrate is oxidized. reciprocal plots obtained for the enzyme reaction data. Increasing coenzyme concentration will increase the Each of the reversible types of inhibition are unique velocity of an enzymatic reaction in a manner syn- with respect to effects on the Vmax and Km parameters onymous with increasing substrate ­ concentration. of enzymatic reactions (Figure 8.4).  Enzyme Kinetics 233 For competitive inhibition, the primary effect is on the interaction of the enzyme with the substrate. Although the Km is a constant for each enzyme–­ substrate pair and cannot be altered, and because the amount of substrate needed to achieve a partic- ular velocity is higher in the presence of a competing inhibitor, the Km appears to increase with the presence of the inhibitor. The effect of the inhibitor can be over- come by adding excess substrate to bind the enzyme. The amount of the inhibitor will become negligible Vmax by comparison, and the reaction will reach the same maximum velocity as an uninhibited reaction. The substrate and inhibitor may both be bound Km to an enzyme in a noncompetitive inhibition. The [S] inhibitor may bind the E form or associate with the A ES form and inhibit the formation of product with the affinity of the inhibitor for E and ES being the same. Thus, for noncompetitive inhibition, the max- imum reaction velocity will not be achieved, and the value measured will be decreased. Inhibitor binding does not influence substrate binding, so the value of Km is unchanged. Mixed type noncompetitive inhibi- tors have the potential to bind both free E and the ES complex with different affinities so changes in both the apparent Vmax and the apparent Km values may be measurable. Vmax Because uncompetitive inhibition requires the formation of an ES complex, increasing substrate concentration can also effectively increase inhibi- tion. Therefore, maximum velocity equal to that of an Km uninhibited reaction cannot be achieved, and the Km [S] B appears to be decreased from the diminished func- tional significance of the ES complex. Irreversible inhibitors are also known and are bet- ter called inactivators. These compounds may bind to proteins and in a time-dependent manner react with an amino acid side chain in the protein. Loss of enzyme activity may result from a reaction at the active site or at another site that causes a structural change in the protein. Such inactivators typically bind the enzyme independent of substrate, so increasing Vmax the concentration of substrate will not prevent or reverse inactivation. Measurement of Enzyme C Km [S] Activity Enzymes are usually present in very small quanti- Figure 8.4 Normal Lineweaver-Burk plot (solid line) compared with each type of enzyme inhibition (dotted ties in biologic fluids and are often difficult to isolate line). (A) Competitive inhibition Vmax unaltered; Km appears from similar compounds, so a convenient method of increased. (B) Noncompetitive inhibition Vmax decreased, enzyme quantification is needed for the measurement Km unchanged. (C) Uncompetitive inhibition Vmax decreased; of catalytic activity, which is then related to concentra- Km appears decreased. tion. Common methods might photometrically mea- © Wolters Kluwer. sure an increase in product concentration, a decrease 234 Chapter 8 Enzymes in substrate concentration, a decrease in coenzyme The temperature should be constant within ±0.1°C concentration, or an increase in the concentration of throughout the assay at a temperature at which the an altered coenzyme. enzyme is active (usually 25°C, 30°C, or 37°C). When all substrates and any coenzyme are pres- During the progress of the reaction, the analysis ent in excess in an enzymatic reaction, the amount of time must be carefully designed and selected. When substrate or coenzyme used or the amount of prod- the enzyme is initially introduced to the substrate to uct or altered coenzyme formed will depend only start the reaction, the high substrate concentration on the amount of enzyme present to catalyze the steadily combines with available enzyme, driving reaction. Using enzyme-catalyzed reactions to mea- the forward reaction to product formation. As the sure enzyme concentrations, therefore, is always per- enzyme becomes effectively saturated, the rates of formed in zero-order kinetics, with the substrate in product formation, release of enzyme, and recom- sufficient excess to ensure that no more than 20% of bination with more substrate reach an equilibrium the available substrate is converted to product. Any such that the net reaction proceeds linearly. After coenzymes also must be in excess. When these con- a time, usually 6 to 8 minutes after reaction initia- ditions are met, the only factor affecting the rate of tion, the reaction rate decreases as the substrate is the reaction will be the concentration of the enzyme depleted. For some enzyme reactions, the amount in the specimen being analyzed. of product present may be sufficient to bind to the NADH is a coenzyme frequently measured in the enzyme in its active site and inhibit the reaction with laboratory. NADH absorbs light at 340 nm, whereas the substrate. Hence, meaningful measurement of NAD+ does not, and a change in absorbance at enzyme activity is performed during the linear phase 340 nm is easily measured. Such reactions follow the of the reaction. change of available NADH at 340 nm, with the rate One of two general methods may be used to mea- calculated using the molar absorptivity (6.22 × 103 sure the extent of an enzymatic reaction: (1) fixed- liter ∙ mol−1 cm−1) of NADH, and lastly conversion to time and (2) continuous-monitoring or kinetic assay. units of enzyme activity. NAD+ or NADH is often con- In the fixed-time method, the reactants are combined, venient as a reagent for a coupled enzyme assay when the reaction proceeds for a designated time, the reac- neither NAD+ nor NADH is a coenzyme for the ini- tion is stopped (usually by inactivating the enzyme tial reaction. In coupled enzyme assays, one or more with a weak acid), and a measurement is made of the enzymes are added in excess as reagents and multi- amount of reaction that has occurred. The reaction is ple reactions are catalyzed. After the enzyme under designed to be linear over the reaction time, achieved analysis catalyzes its specific reaction, a product of by first evaluating the reaction for various times aim- that reaction becomes the substrate for the reaction ing to find a suitable time for adequate measurement catalyzed by an intermediate auxiliary enzyme. The of the reaction, typically by substrate loss or product product of the intermediate reaction becomes the appearance. The greater the reaction measurement, substrate for the final reaction, which is catalyzed the more enzyme is present. by an indicator enzyme and commonly involves In continuous-monitoring or kinetic assays, the conversion of NAD+ to NADH or vice versa. For multiple measurements, usually of absorbance many assays, the second enzymatic reaction provides change, are made during the reaction, either at spe- an indicator, in which case the auxiliary enzyme is cific time intervals (usually every 30 or 60 seconds) also the indicator enzyme. or continuously by a continuous-recording spectro- When performing an enzyme quantification in photometer. Collection of data at specific time inter- zero-order kinetics, inhibitors must not be present, vals is essentially a series of stopped-time assays to and other variables that may influence the rate of the construct a linear plot of the reaction. These assays reaction must be carefully controlled. All substrates are advantageous over fixed-time methods because and cofactors must be present in excess, so substrate the linearity of the reaction may be more adequately concentration does not change the kinetics to first or verified. If absorbance is measured at intervals, sev- second order. (Second-order kinetic reactions occur eral data points are necessary to increase the accu- when concentrations of two different substrates are racy of assessed linearity. Continuous measurements needed to produce a product, so both substrates affect are preferred because any deviation from linearity is the rate of reaction. Second-order reactions are not readily observable. used in the clinical laboratory and therefore not dis- The most common cause of deviation from linear- cussed in this chapter.) A constant pH should be main- ity occurs when the amount of enzyme is so elevated tained by means of an appropriate buffer solution. that all substrate is used rapidly in the reaction time,  Enzyme Kinetics 235 effectively making the linear phase for the reaction enzyme present as mass or concentration. The rela- quite short. With continuous monitoring, the labora- tionship between enzyme activity and enzyme quan- torian may observe a sudden decrease in the reaction tity is generally linear but should be verified with the rate (deviation from zero-order kinetics) of a particu- conditions used for each enzyme under study. The lar determination and may repeat the determination mass of enzymes may also be determined by elec- using less patient sample. When possible, the analysis trophoretic techniques, which provide resolution of should be repeated using less patient sample rather isoenzymes and isoforms. Immunoassay methodol- than diluting the sample in order to eliminate the ogies that quantify enzyme concentration by mass possibility of the diluent interfering with the reaction. are also available and are routinely used for quan- The result obtained will still be multiplied by any tification of some enzymes, such as creatine kinase dilutional factor. (Sample dilution with saline may isoenzyme CK-MB. Immunoassays may overestimate be necessary to minimize negative effects in analysis active enzyme due to possible cross-reactivity with caused by hemolysis or lipemia). Less sample should inactive enzymes, such as zymogens, inactive isoen- catalyze less reaction over the same time period to zymes, macroenzymes, or partially digested enzyme. provide for a longer reaction time being observed Ensuring the accuracy of enzyme measurements as linear. Enzyme activity measurements may not be has long been a concern of laboratorians. The Clin- accurate if storage conditions compromise integrity ical Laboratory Improvement Amendment of 1988 of the protein, if enzyme inhibitors are present, or if (CLIA 88) has established guidelines for quality required cofactors are not present. control and proficiency testing for all laboratories. Problems with quality control materials for enzyme Calculation of Enzyme Activity testing have been a significant issue. Differences When enzymes are quantified relative to their activity between clinical specimens and control samples rather than a direct measurement of concentration, include species of origin of the enzyme, integrity the units used to report enzyme levels are activity of the molecular species, isoenzyme forms, matrix units. The definition for the activity unit must con- of the solution, addition of preservatives, and lyo- sider conditions that may alter results (e.g., pH, tem- philization processes. Many studies have been con- perature, substrate). Historically, specific method ducted to ensure accurate enzyme measurements developers frequently established their own units and good quality control materials.4 for reporting results and often named the units after themselves (i.e., Bodansky and King units). To stan- Enzymes as Reagents dardize the system of reporting quantitative results, Enzymes may be used as reagents to measure many the EC defined the international unit (IU) as the nonenzymatic constituents in serum. For example, amount of enzyme that will catalyze the reaction of glucose, cholesterol, and uric acid are examples of 1 μmol of substrate per minute while also includ- biomolecules frequently quantified by means of ing descriptions of the specified conditions of tem- enzymatic reactions that measure the concentration perature, pH, substrates, and activators used in the of the analyte by following the reaction catalyzed reaction. Such details are provided to enable other by an enzyme. Enzymes are also used as reagents laboratorians to replicate the results. Because speci- in methods to quantify analytes that are substrates fied conditions may vary among laboratories, refer- for the corresponding enzyme. One example, lactate ence ranges are still often laboratory specific. Enzyme dehydrogenase (LD), may be a reagent when lactate concentration is usually expressed in units per liter or pyruvate concentrations are evaluated. For such (IU/L). The unit of enzyme activity recognized by the methods, the enzyme is added in excess in a quan- International System of Units (Système International tity sufficient to provide a complete reaction in a d’Unités [SI]) is the katal (mol/s). The mole is the unit short period. for substrate concentration, and the unit of time is The nature of the enzyme being used may allow the second. Enzyme concentration is then expressed as katals per liter (kat/L) (1.0 IU = 17 nkat). the development of novel approaches for the anal- ysis. Immobilized enzymes are chemically linked to adsorbents, such as agarose or certain types of Measurement of Enzyme Mass cellulose, via covalent bonds often through azide Understanding these units of enzyme activity also groups, diazo, and triazine. When substrate is requires knowledge of the amount of enzyme avail- passed through the preparation, the product is able in the reaction, more specifically, the amount of retrieved and analyzed, and the enzyme is present 236 Chapter 8 Enzymes and free to react with more substrate. Immobilized predominant physiologic function occurs in mus- enzymes are convenient for batch analyses and are cle cells, where it is involved in the storage of high-­ more stable than enzymes in a solution. Enzymes are energy creatine phosphate. Every contraction cycle also commonly used as reagents in competitive and of muscle results in creatine phosphate use with the noncompetitive immunoassays, such as those used production of ATP. This results in relatively constant to measure human immunodeficiency virus (HIV) levels of muscle ATP. The reversible reaction cata- antibodies, therapeutic drugs, and cancer antigens. lyzed by CK is shown in Equation 8.4. Enzymes often are covalently linked (conjugated) to CK a secondary antibody to reflect the amount of the Creatine  ATP Creatine phosphate  ADP primary antigen. Commonly used enzymes include horseradish peroxidase, alkaline phosphatase (ALP), (Eq. 8.4) glucose-6-phosphate dehydrogenase (G6PD), and β-galactosidase. The enzyme in these assays func- Tissue Source tions as an indicator that reflects either the presence CK is widely distributed in tissue, with highest activi- or absence of the analyte. ties found in skeletal muscle, heart muscle, and brain tissue. CK is present in much smaller quantities in other tissues, including the bladder, placenta, gastro- Enzymes of Clinical intestinal tract, thyroid, uterus, kidney, lung, prostate, Significance spleen, liver, and pancreas. Table 8.2 lists the major enzymes of clinical signifi- Diagnostic Significance cance, including their systematic names and clinical significance. The most frequently analyzed clinical Due to the high concentrations of CK in muscle tissue, enzymes are discussed in this chapter with respect to plasma CK levels are frequently elevated in disorders tissue source, diagnostic significance, assay method, of cardiac and skeletal muscle (myocardial infarction source of error, and reference range. [MI], rhabdomyolysis, and muscular dystrophy). The CK level is considered a sensitive indicator of acute myocardial infarction (AMI) and muscular dystrophy, Creatine Kinase particularly the Duchenne type. Extreme elevations of CK is an enzyme with a molecular weight of approx- CK occur in Duchenne-type muscular dystrophy, with imately 82,000 that is generally associated with ATP values reaching 50 to 100 times the upper limit of regeneration in contractile or transport systems. Its normal (ULN). Although total CK levels are s­ ensitive CASE STUDY 8.1, PART 2 Remember Carl. At the doctor’s office, an electrocardiogram revealed changes from one per- formed 6 months earlier. The results of the patient’s blood work are as follows: Test Result Reference Range CK 129 U/L (30–60) CK-MB 4% ( LD-2 © Tony Wear/Shutterstock. AST 35 U/L (5–30) TnT 12 ng/L (