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13 Enzymes C H A P T E R KAMISHA L. JOHNSON-DAVIS...

13 Enzymes C H A P T E R KAMISHA L. JOHNSON-DAVIS CHAPTER OUTLINE u GENERAL PROPERTIES AND DEFINITIONS Lactate Dehydrogenase u ENZYME CLASSIFICATION AND Aspartate Aminotransferase NOMENCLATURE Alanine Aminotransferase u ENZYME KINETICS Alkaline Phosphatase Catalytic Mechanism of Enzymes Acid Phosphatase Factors That Influence Enzymatic γ-Glutamyltransferase Reactions Amylase Measurement of Enzyme Activity Lipase Calculation of Enzyme Activity Glucose-6-Phosphate Dehydrogenase Measurement of Enzyme Mass Drug-Metabolizing Enzymes Enzymes as Reagents u QUESTIONS u ENZYMES OF CLINICAL SIGNIFICANCE u REFERENCES Creatine Kinase Chapter Objectives various disorders, including cardiac, hepatic, bone, and Upon completion of this chapter, the clinical muscle, malignancies, and acute pancreatitis. ­laboratorian should be able to do the following: Discuss the tissue sources, diagnostic significance, and assays, including sources of error, for the following Define the term enzyme, including physical composition enzymes: creatine kinase, lactate dehydrogenase, aspar- and structure. tate a­minotransferase, alanine aminotransferase, alkaline Classify enzymes according to the International Union of phosphatase, acid phosphatase, γ-glutamyltransferase, Biochemistry. amylase, lipase, cholinesterase, and glucose-6-phosphate Discuss the different factors affecting the rate of an dehydrogenase. ­enzymatic reaction. Evaluate patient serum enzyme levels in relation to disease Explain enzyme kinetics including zero-order and states. ­first-order kinetics. Discuss the clinical importance for detecting Explain why the measurement of serum enzyme levels is ­macroenzymes. clinically useful. Discuss the role of enzymes in drug metabolism. Discuss which enzymes are useful in the diagnosis of Key Terms Hydrolase International unit (IU) Activation energy Isoenzyme Activators Isoform Apoenzyme Kinetic assay Coenzyme LD flipped pattern Cofactor Michaelis-Menten constant Enzyme Oxidoreductase Enzyme–substrate (ES) complex Transferase First-order kinetics Zero-order kinetics Holoenzyme Zymogen 262 18698_ch13_p262-291.indd 262 10/11/12 11:20 AM  CHAPTER 13 n ENZYMES 263 Enzymes are specific biologic proteins that catalyze ­ inucleotide (NAD). When bound tightly to the enzyme, d biochemical reactions without altering the equilibrium the coenzyme is called a prosthetic group. The enzyme point of the reaction or being consumed or changed portion (apoenzyme), with its respective coenzyme, in composition. The other substances in the reaction forms a complete and active system, a holoenzyme. are converted to products. The catalyzed reactions are Some enzymes, mostly digestive enzymes, are origi- frequently specific and essential to physiologic func- nally secreted from the organ of production in a struc- tions, such as the hydration of carbon dioxide, nerve turally inactive form, called a proenzyme or zymogen. conduction, muscle contraction, nutrient degradation, Other enzymes later alter the structure of the proenzyme and energy use. Found in all body tissues, enzymes to make active sites available by hydrolyzing specific frequently appear in the serum following cellular injury amino acid residues. This mechanism prevents digestive or, sometimes, in smaller amounts, from degraded cells. enzymes from digesting their place of synthesis. Certain enzymes, such as those that facilitate coagula- tion, are specific to plasma and, therefore, are pres- Enzyme Classification and ent in significant concentrations in plasma. Plasma or Nomenclature serum enzyme levels are often useful in the diagnosis To standardize enzyme nomenclature, the Enzyme of particular diseases or physiologic abnormalities. This Commission (EC) of the IUB adopted a classification chapter discusses the general properties and principles system in 1961; the standards were revised in 1972 and of enzymes, aspects relating to the clinical diagnostic 1978. The IUB system assigns a systematic name to each significance of specific physiologic enzymes, and assay enzyme, defining the substrate acted on, the reaction methods for those enzymes. catalyzed, and, possibly, the name of any coenzyme involved in the reaction. Because many systematic names General Properties and Definitions are lengthy, a more usable, trivial, recommended name is Enzymes catalyze many specific physiologic reactions. also assigned by the IUB system.1 These reactions are facilitated by the enzyme structure In addition to naming enzymes, the IUB system iden- and several other factors. As a protein, each enzyme con- tifies each enzyme by an EC numerical code containing tains a specific amino acid sequence (primary structure), four digits separated by decimal points. The first digit with the resultant polypeptide chains twisting (second- places the enzyme in one of the following six classes: ary structure), which then folds (tertiary structure) and 1. Oxidoreductases. Catalyze an oxidation–reduction results in structural cavities. If an enzyme contains more reaction between two substrates than one polypeptide unit, the quaternary structure refers 2. Transferases. Catalyze the transfer of a group other to the spatial relationships between the subunits. Each than hydrogen from one substrate to another enzyme contains an active site, often a water-free cav- 3. Hydrolases. Catalyze hydrolysis of various bonds ity, where the substance on which the enzyme acts (the 4. Lyases. Catalyze removal of groups from substrates substrate) interacts with particular charged amino acid without hydrolysis; the product contains double residues. An allosteric site—a cavity other than the active bonds site—may bind regulator molecules and, thereby, be sig- 5. Isomerases. Catalyze the interconversion of geomet- nificant to the basic enzyme structure. ric, optical, or positional isomers Even though a particular enzyme maintains the same 6. Ligases. Catalyze the joining of two substrate mol- catalytic function throughout the body, that enzyme may ecules, coupled with breaking of the pyrophosphate exist in different forms within the same individual. The bond in adenosine triphosphate (ATP) or a similar different forms may be differentiated from each other compound based on certain physical properties, such as electropho- retic mobility, solubility, or resistance to inactivation. The second and third digits of the EC code number The term isoenzyme is generally used when discuss- represent the subclass and subsubclass of the enzyme, ing such enzymes; however, the International Union of respectively, divisions that are made according to criteria Biochemistry (IUB) suggests restricting this term to mul- specific to the enzymes in the class. The final number is tiple forms of genetic origin. An isoform results when the serial number specific to each enzyme in a subsub- an enzyme is subject to posttranslational modifications. class. Table 13-1 provides the EC code numbers, as well Isoenzymes and isoforms contribute to heterogeneity in as the systematic and recommended names, for enzymes properties and function of enzymes. frequently measured in the clinical laboratory. In addition to the basic enzyme structure, a nonpro- Table 13-1 also lists common and standard abbre- tein molecule, called a cofactor, may be necessary for viations for commonly analyzed enzymes. Without IUB enzyme activity. Inorganic cofactors, such as chloride recommendation, capital letters have been used as a or magnesium ions, are called activators. A coenzyme ­convenience to identify enzymes. The common abbre- is an organic cofactor, such as nicotinamide adenine viations, sometimes developed from previously accepted 18698_ch13_p262-291.indd 263 10/11/12 11:20 AM  264 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES Table 13-1 Classification Of Frequently Quantitated Enzymes Recommended Common Standard EC Code Class Name Abbreviation Abbreviation No. Systematic Name Oxidoreductases Lactate LD LD 1.1.1.27 l-Lactate:NAD+ dehydrogenase oxidoreductase Glucose-6-phosphate G-6-PDH G-6-PD 1.1.1.49 d-Glucose-6- dehydrogenase phosphate:NADP+ 1-oxidoreductase Glutamate GLD GLD 1.4.1.3 l-glutamate:NAD(P) dehydrogenase oxidoreductase, deaminase Transferases Aspartate GOT (glutamate AST 2.6.1.1 l-Aspartate:2- aminotransferase oxaloacetate oxaglutarate transaminase) aminotransferase Alanine GPT (glutamate ALT 2.6.1.2 l-Alanine:2-oxaglutarate aminotransferase transaminase) aminotransferase Creatine kinase CPK (creatine CK 2.7.3.2 ATP:creatine phosphokinase) N-phosphotransferase γ-Glutamyltransferase GGTP GGT 2.3.2.2 (5-Glutamyl)peptide: amino acid-5-glutamyl- transferase 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 phospho- hydrolase (alkaline ­optimum) Acid phosphatase ACP ACP 3.1.3.2 Orthophosphoric monoester phosphohy- drolase (acid optimum) α-Amylase AMS AMY 3.2.1.1 1,4-d-Glucan glucanohydrolase Cholinesterase PCHE CHE 3.1.1.8 Acylcholine acylhydrolase Chymotrypsin CHY CHY 3.4.21.1 Chymotrypsin Elastase-1 E1 E1 3.4.21.36 Elastase 5-Nucleotidase NTP NTP 3.1.3.5 5′-Ribonucleotide phosphohydrolase Triacylglycerol lipase LPS 3.1.1.3 Triacylglycerol acylhydrolase Trypsin TRY TRY 3.4.21.4 Trypsin Lyases Aldolase ALD ALD 4.1.2.13 d-d-Fructose-1,6-bisdi- phosphated-glyceral- dehyde-3-phosphate- lyase Isomerases Triosephosphate TPI TPI 5.3.1.1 Triose-phosphate isomerase isomerase Ligase Glutathione GSH-S GSH-S 6.3.2.3 Glutathione synthase synthetase Adapted from Competence Assurance, ASMT. Enzymology, an Educational Program. Bethesda, MD: RMI Corporation; 1980. 18698_ch13_p262-291.indd 264 10/11/12 11:20 AM  CHAPTER 13 n ENZYMES 265 names for the enzymes, were used until the standard The general relationship among the enzyme, substrate, abbreviations listed in the table were developed.2,3 These and product may be represented as follows: standard abbreviations are used in the United States and E + S → ES → E + P (Eq. 13-1) are used later in this chapter to indicate specific enzymes. where E is the enzyme, S is the substrate, ES is the Enzyme Kinetics enzyme–substrate complex, and P is the product. The ES complex is a physical binding of a substrate Catalytic Mechanism of Enzymes to the active site of an enzyme. The structural arrange- A chemical reaction may occur spontaneously if the ment of amino acid residues within the enzyme makes free energy or available kinetic energy is higher for the the three-dimensional active site available. At times, the reactants than for the products. The reaction then pro- binding of ligand drives active site rearrangement. The ceeds toward the lower energy if a sufficient number of transition state for the ES complex has a lower energy of the reactant molecules possess enough excess energy activation than the transition state of S alone, so that the to break their chemical bonds and collide to form new reaction proceeds after the complex is formed. An actual bonds. The excess energy, called activation energy, is reaction may involve several substrates and products. the energy required to raise all molecules in 1 mol of Different enzymes are specific to substrates in differ- a compound at a certain temperature to the transition ent extents or respects. Certain enzymes exhibit absolute state at the peak of the energy barrier. At the transition specificity, meaning that the enzyme combines with only state, each molecule is equally likely to either participate one substrate and catalyzes only the one correspond- in product formation or remain an unreacted molecule. ing reaction. Other enzymes are group specific because Reactants possessing enough energy to overcome the they combine with all substrates containing a particular energy barrier participate in product formation. chemical group, such as a phosphate ester. Still other One way to provide more energy for a reaction is enzymes are specific to chemical bonds and thereby to increase the temperature and thus increase intermo- exhibit bond specificity. lecular collisions; however, this does not normally occur Stereoisometric specificity refers to enzymes that pre- physiologically. Enzymes catalyze physiologic reactions dominantly combine with only one optical isomer of by lowering the activation energy level that the reac- a certain compound. In addition, an enzyme may bind tants (substrates) must reach for the reaction to occur more than one molecule of substrate, and this may (Fig. 13-1). The reaction may then occur more readily to occur in a cooperative fashion. Binding of one substrate a state of equilibrium in which there is no net forward or molecule, therefore, may facilitate binding of additional reverse reaction, even though the equilibrium constant substrate molecules. of the reaction is not altered. The extent to which the reaction progresses depends on the number of substrate Factors That Influence Enzymatic Reactions molecules that pass the energy barrier. Substrate Concentration The rate at which an enzymatic reaction proceeds and whether the forward or reverse reaction occurs depend on several reaction conditions. One major influence on enzymatic reactions is substrate concentration. In 1913, Michaelis and Menten hypothesized the role of substrate concentration in the formation of the enzyme–substrate (ES) complex. According to their hypothesis, represented in Figure 13-2, the substrate readily binds to free enzyme at a low substrate con- centration. With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily increases as more substrate is added. The reaction is following first-order kinetics because the reaction rate is directly proportional to substrate concentration. Eventually, however, the substrate concentration is high enough to saturate all available enzyme, and the reaction velocity reaches its maximum. When the product is formed, the resultant free enzyme immediately combines with FIGURE 13-1 Energy vs. progression of reaction, indicating the energy barrier that the substrate must surpass to react with and excess free substrate. The reaction is in zero-order without enzyme catalysis. The enzyme considerably reduces the kinetics, and the reaction rate depends only on enzyme free energy needed to activate the reaction. concentration. 18698_ch13_p262-291.indd 265 10/11/12 11:20 AM  266 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES V Vmax Km FIGURE 13-2 Michaelis-Menten curve of velocity vs. substrate con- Km centration for enzymatic reaction. Km is the substrate concentration FIGURE 13-3 Lineweaver-Burk transformation of Michaelis-Menten at which the reaction velocity is half of the maximum level. curve. Vmax is the reciprocal of the x-intercept of the straight line. Km is the negative reciprocal of the x-intercept of the same line. The Michaelis-Menten constant (Km), derived from the theory of Michaelis and Menten, is a constant for a specific enzyme and substrate under defined reac- taken of both the substrate concentration and the velocity tion conditions and is an expression of the relationship of an enzymatic reaction. The equation becomes between the velocity of an enzymatic reaction and sub- 1 ​​ 1 ​Km 1 strate concentration. The assumptions are made that ​ __ ​= _____ ​  ​___ ​   ​+ _____ ​   ​ (Eq. 13-3) V ​Vmax ​ ​ [S] ​Vmax ​ ​ equilibrium among E, S, ES, and P is established rapidly and that the E + P → ES reaction is negligible. The rate- Enzyme Concentration limiting step is the formation of product and enzyme Because enzymes catalyze physiologic reactions, the from the ES complex. Then, maximum velocity is fixed, enzyme concentration affects the rate of the catalyzed and the reaction rate is a function of only the enzyme reaction. As long as the substrate concentration exceeds concentration. As designated in Figure 13-2, Km is spe- the enzyme concentration, the velocity of the reaction is cifically the substrate concentration at which the enzyme proportional to the enzyme concentration. The higher yields half the possible maximum velocity. Therefore, Km the enzyme level, the faster the reaction will proceed indicates the amount of substrate needed for a particular because more enzyme is present to bind with the sub- enzymatic reaction. strate. The Michaelis-Menten hypothesis of the relationship between reaction velocity and substrate concentration pH can be represented mathematically as follows: Enzymes are proteins that carry net molecular charges. ​Vmax ​ ​[S] Changes in pH may denature an enzyme or influence its V = ________ ​   ​ (Eq. 13-2) ionic state, resulting in structural changes or a change ​K​m​+ [S] in the charge of an amino acid residue in the active site. Hence, each enzyme operates within a specific pH range where V is the measured velocity of reaction, Vmax is the and maximally at a specific pH. Most physiologic enzy- maximum velocity, [S] is the substrate concentration, matic reactions occur in the pH range of 7.0 to 8.0, but and Km is the Michaelis-Menten constant of enzyme for some enzymes are active in wider pH ranges than others. a specific substrate. In the laboratory, the pH for a reaction is carefully con- Theoretically, Vmax and then Km could be determined trolled at the optimal pH by means of appropriate buffer from the plot in Figure 13-2. However, Vmax is difficult to solutions. determine from the hyperbolic plot and often not actually achieved in enzymatic reactions because enzymes may not Temperature function optimally in the presence of excessive substrate. Increasing the temperature usually increases the rate A more accurate and convenient determination of Vmax of a chemical reaction by increasing the movement of and Km may be made through a Lineweaver-Burk plot, a molecules, the rate at which intermolecular collisions double-reciprocal plot of the Michaelis-Menten constant, occur, and the energy available for the reaction. This is which yields a straight line (Fig. 13-3). The reciprocal is the case with enzymatic reactions until the temperature 18698_ch13_p262-291.indd 266 10/11/12 11:20 AM  CHAPTER 13 n ENZYMES 267 is high enough to denature the protein composition of Inhibitors the enzyme. For each 10° increase in temperature, the Enzymatic reactions may not progress normally if a par- rate of the reaction will approximately double until, of ticular substance, an inhibitor, interferes with the reac- course, the protein is denatured. tion. Competitive inhibitors physically bind to the active Each enzyme functions optimally at a particular tem- site of an enzyme and compete with the substrate for the perature, which is influenced by other reaction variables, active site. With a substrate concentration significantly especially the total time for the reaction. The optimal higher than the concentration of the inhibitor, the inhi- temperature is usually close to that of the physiologic bition is reversible because the substrate is more likely environment of the enzyme; however, some denaturation than the inhibitor to bind the active site and the enzyme may occur at the human physiologic temperature of has not been destroyed. 37°C. The rate of denaturation increases as the tempera- A noncompetitive inhibitor binds an enzyme at a place ture increases and is usually significant at 40°C to 50°C. other than the active site and may be reversible in that Because low temperatures render enzymes reversibly some naturally present metabolic substances combine inactive, many serum or plasma specimens for enzyme reversibly with certain enzymes. Noncompetitive inhi- measurement are refrigerated or frozen to prevent activ- bition also may be irreversible if the inhibitor destroys ity loss until analysis. Storage procedures may vary from part of the enzyme involved in catalytic activity. Because enzyme to enzyme because of individual stability charac- the inhibitor binds the enzyme independently from the teristics. Repeated freezing and thawing, however, tends substrate, increasing substrate concentration does not to denature protein and should be avoided. reverse the inhibition. Because of their temperature sensitivity, enzymes Uncompetitive inhibition is another kind of inhibition in should be analyzed under strictly controlled temperature which the inhibitor binds to the ES complex—increasing conditions. Incubation temperatures should be accurate substrate concentration results in more ES complexes within ±0.1°C. Laboratories usually attempt to establish to which the inhibitor binds and, thereby, increases the an analysis temperature for routine enzyme measure- inhibition. The enzyme–substrate–inhibitor complex ment of 25°C, 30°C, or 37°C. Attempts to establish a does not yield product. universal temperature for enzyme analysis have been Each of the three kinds of inhibition is unique with unsuccessful, and therefore, reference ranges for enzyme respect to effects on the Vmax and Km of enzymatic reac- levels may vary significantly among laboratories. In the tions (Fig. 13-4). In competitive inhibition, the effect United States, however, 37°C is most commonly used. of the inhibitor can be counteracted by adding excess Cofactors substrate to bind the enzyme. The amount of the inhibi- Cofactors are nonprotein entities that must bind to tor is then negligible by comparison, and the reaction particular enzymes before a reaction occurs. Common will proceed at a slower rate but to the same maximum activators (inorganic cofactors) are metallic (Ca2+, Fe2+, velocity as an uninhibited reaction. Km is a constant for Mg2+, Mn2+, Zn2+, and K+) and nonmetallic (Br- and each enzyme and cannot be altered. However, because Cl–). The activator may be essential for the reaction or the amount of substrate needed to achieve a particular may only enhance the reaction rate in proportion with velocity is higher in the presence of a competing inhibi- concentration to the point at which the excess activa- tor, Km appears to increase when exhibiting the effect of tor begins to inhibit the reaction. Activators function by the inhibitor. alternating the spatial configuration of the enzyme for The substrate and inhibitor, commonly a metallic proper substrate binding, linking substrate to the enzyme ion, may bind an enzyme simultaneously in noncom- or coenzyme, or undergoing oxidation or reduction. petitive inhibition. The inhibitor may inactivate either Some common coenzymes (organic cofactors) are an ES complex or just the enzyme by causing struc- nucleotide phosphates and vitamins. Coenzymes serve as tural changes in the enzyme. Even if the inhibitor binds second substrates for enzymatic reactions. When bound reversibly and does not inactivate the enzyme, the pres- tightly to the enzyme, coenzymes are called prosthetic ence of the inhibitor when it is bound to the enzyme groups. For example, NAD as a cofactor may be reduced slows the rate of the reaction. Thus, for noncompetitive to nicotinamide adenine dinucleotide phosphate (NADP) inhibition, the maximum reaction velocity cannot be in a reaction in which the primary substrate is oxidized. achieved. Increasing substrate levels have no influence Increasing coenzyme concentration will increase the on the binding of a noncompetitive inhibitor, so that Km velocity of an enzymatic reaction in a manner synony- is unchanged. mous with increasing substrate concentration. When Because uncompetitive inhibition requires the forma- quantitating an enzyme that requires a particular cofac- tion of an ES complex, increasing substrate concentra- tor, that cofactor should always be provided in excess so tion increases inhibition. Therefore, maximum ­velocity that the extent of the reaction does not depend on the equal to that of an uninhibited reaction cannot be concentration of the cofactor. achieved, and Km appears to be decreased. 18698_ch13_p262-291.indd 267 10/11/12 11:20 AM  268 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES Vmax Vmax Vmax Km Km Km [S] [S] [S] FIGURE 13-4 Normal Lineweaver-Burk plot (solid line) compared with each type of enzyme inhibition (dotted line). (A) Competitive inhibi- tion Vmax unaltered; Km appears increased. (B) Noncompetitive inhibition Vmax decreased; Km unchanged. (C) Uncompetitive inhibition Vmax decreased; Km appears decreased. Measurement of Enzyme Activity the substrate on which an intermediate auxiliary enzyme acts. A product of the intermediate reaction becomes the Because enzymes are usually present in very small quan- substrate for the final reaction, which is catalyzed by an tities in biologic fluids and often difficult to isolate from indicator enzyme and commonly involves the conversion similar compounds, a convenient method of enzyme of NAD to NADH or vice versa. quantitation is measurement of catalytic activity. Activity When performing an enzyme quantitation in zero- is then related to concentration. Common methods order kinetics, inhibitors must be lacking and other might photometrically measure an increase in product variables that may influence the rate of the reaction must concentration, a decrease in substrate concentration, a be carefully controlled. A constant pH should be main- decrease in coenzyme concentration, or an increase in tained by means of an appropriate buffer solution. The the concentration of an altered coenzyme. temperature should be constant within ±0.1°C through- If the amount of substrate and any coenzyme is in out the assay at a temperature at which the enzyme is excess in an enzymatic reaction, the amount of substrate active (usually, 25°C, 30°C, or 37°C). or coenzyme used, or product or altered coenzyme During the progress of the reaction, the period for formed, will depend only on the amount of enzyme the analysis also must be carefully selected. When the present to catalyze the reaction. Enzyme concentrations, enzyme is initially introduced to the reactants and the therefore, are always performed in zero-order kinetics, excess substrate is steadily combining with the avail- with the substrate in sufficient excess to ensure that no able enzyme, the reaction rate rises. After the enzyme is more than 20% of the available substrate is converted to saturated, the rates of product formation, release of the product. Any coenzyme also must be in excess. NADH enzyme, and recombination with more substrate proceed is a coenzyme frequently measured in the laboratory. linearly. After some time, usually 6 to 8 minutes after NADH absorbs light at 340 nm, whereas NAD does reaction initiation, the reaction rate decreases as the not, and a change in absorbance at 340 nm is easily substrate is depleted, the reverse reaction occurs appre- measured. ciably, and the product begins to inhibit the reaction. In specific laboratory methodologies, substances Hence, enzyme quantitations must be performed during other than substrate or coenzyme are necessary and the linear phase of the reaction. must be present in excess. NAD or NADH is often con- One of two general methods may be used to measure venient as a reagent for a coupled-enzyme assay when the extent of an enzymatic reaction: (1) fixed-time and neither NAD nor NADH is a coenzyme for the reaction. (2) continuous-monitoring or kinetic assay. In the fixed- In other coupled-enzyme assays, more than one enzyme time method, the reactants are combined, the reaction is added in excess as a reagent and multiple reactions proceeds for a designated time, the reaction is stopped are catalyzed. After the enzyme under analysis catalyzes (usually by inactivating the enzyme with a weak acid), its specific reaction, a product of that reaction becomes and a measurement of the amount of reaction that has 18698_ch13_p262-291.indd 268 10/11/12 11:20 AM  CHAPTER 13 n ENZYMES 269 occurred is made. The reaction is assumed to be linear of Units (Système International d’Unités [SI]) is the over the reaction time; the larger the reaction, the more katal (mol/s). The mole is the unit for substrate concen- the enzyme present. tration, and the unit of time is the second. Enzyme con- In continuous-monitoring or kinetic assays, multiple centration is then expressed as katals per liter (kat/L) measurements, usually of absorbance change, are made (1.0 IU = 17 nkat). during the reaction, either at specific time intervals When enzymes are quantitated by measuring the (usually every 30 or 60 seconds) or continuously by a increase or decrease of NADH at 340 nm, the molar continuous-recording spectrophotometer. These assays absorptivity (6.22 × 103 mol/L) of NADH is used to cal- are advantageous over fixed-time methods because culate enzyme activity. the linearity of the reaction may be more adequately verified. If absorbance is measured at intervals, several Measurement of Enzyme Mass data points are necessary to increase the accuracy of linearity assessment. Continuous measurements are Immunoassay methodologies that quantify enzyme preferred because any deviation from linearity is readily concentration by mass are also available and are observable. routinely used for quantification of some enzymes, The most common cause of deviation from linearity such as creatine kinase (CK)-MB. Immunoassays may occurs when the enzyme is so elevated that all substrate overestimate active enzyme as a result of possible is used early in the reaction time. For the remainder cross-reactivity with inactive enzymes, such as zymo- of the reaction, the rate change is minimal, with the gens, inactive isoenzymes, macroenzymes, or partially implication that the coenzyme concentration is very digested enzyme. The relationship between enzyme low. With continuous monitoring, the laboratorian may activity and enzyme quantity is generally linear but observe a sudden decrease in the reaction rate (devia- should be determined for each enzyme. Enzymes may tion from zero-order kinetics) of a particular determi- also be determined and quantified by electrophoretic nation and may repeat the determination using less techniques, which provide resolution of isoenzymes patient sample. The decrease in the amount of patient and isoforms. sample operates as a dilution, and the answer obtained Ensuring the accuracy of enzyme measurements may be multiplied by the dilution factor to obtain the has long been a concern of laboratorians. The Clinical final answer. The sample itself is not diluted so that Laboratory Improvement Amendment of 1988 (CLIA the diluent cannot interfere with the reaction. (Sample ’88) has established guidelines for quality control and dilution with saline may be necessary to minimize nega- proficiency testing for all laboratories. Problems with tive effects in analysis caused by hemolysis or lipemia.) quality control materials for enzyme testing have been Enzyme activity measurements may not be accurate if a significant issue. Differences between clinical speci- storage conditions compromise integrity of the protein, mens and control sera include species of origin of the if enzyme inhibitors are present, or if necessary cofac- enzyme, integrity of the molecular species, isoenzyme tors are not present. forms, matrix of the solution, addition of preservatives, and lyophilization processes. Many studies have been Calculation of Enzyme Activity conducted to ensure accurate enzyme measurements and good quality control materials.4 When enzymes are quantified relative to their activity rather than a direct measurement of concentration, the Enzymes as Reagents units used to report enzyme levels are activity units. The definition for the activity unit must consider vari- Enzymes may be used as reagents to measure many ables that may alter results (e.g., pH, temperature, and nonenzymatic constituents in serum. For example, glu- substrate). Historically, specific method developers cose, cholesterol, and uric acid are frequently quantified frequently established their own units for reporting by means of enzymatic reactions, which measure the results and often named the units after themselves (i.e., concentration of the analyte due to the specificity of the Bodansky and King units). To standardize the system enzyme. Enzymes are also used as reagents for methods of reporting quantitative results, the EC defined the to quantify analytes that are substrates for the corre- international unit (IU) as the amount of enzyme that sponding enzyme. One example, lactate dehydrogenase will catalyze the reaction of 1 μmol of substrate per (LD), may be a reagent when lactate or pyruvate concen- minute under specified conditions of temperature, pH, trations are evaluated. For such methods, the enzyme substrates, and activators. Since specified conditions is added in excess in a quantity sufficient to provide a may vary among laboratories, reference values are still complete reaction in a short period. often laboratory specific. Enzyme concentration is Immobilized enzymes are chemically bonded to adsor- usually expressed in units per liter (IU/L). The unit of bents, such as agarose or certain types of cellulose, by enzyme activity recognized by the International System azide groups, diazo, and triazine. The enzymes act as 18698_ch13_p262-291.indd 269 10/11/12 11:20 AM  270 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES recoverable reagents. When substrate is passed through peroxidase, alkaline phosphatase (ALP), glucose-6-phos- the preparation, the product is retrieved and analyzed, phate dehydrogenase (G-6-PD), and β-galactosidase. and the enzyme is present and free to react with more The enzyme in these assays functions as an indicator that substrate. Immobilized enzymes are convenient for reflects either the presence or the absence of the analyte. batch analyses and are more stable than enzymes in a solution. Enzymes are also commonly used as reagents Enzymes of Clinical Significance in competitive and noncompetitive immunoassays, such as those used to measure human immunodeficiency Table 13-2 lists the commonly analyzed enzymes, includ- virus antibodies, therapeutic drugs, and cancer anti- ing their systematic names and clinical significance. Each gens. Commonly used enzymes include horseradish enzyme is discussed in this chapter with respect to tissue Table 13-2 Major Enzymes of Clinical Significance Enzyme Clinical Significance Acid phosphatase (ACP) Prostatic carcinoma Alanine aminotransferase (ALT) Hepatic disorder Aldolase (ALD) Skeletal muscle disorder Alkaline phosphatase (ALP) Hepatic disorder Bone disorder Amylase (AMY) Acute pancreatitis Angiotensin-converting enzyme (ACE) Blood pressure regulation Aspartate aminotransferase (AST) Myocardial infarction Hepatic disorder Skeletal muscle disorder Chymotrypsin (CHY) Chronic pancreatitis insufficiency Creatine kinase (CK) Myocardial infarction Skeletal muscle disorder Elastase-1 (E1) Chronic pancreatitis insufficiency Glucose-6-phosphate dehydrogenase (G-6-PD) Drug-induced hemolytic anemia Glutamate dehydrogenase (GLD) Hepatic disorder γ-Glutamyltransferase (GGT) Hepatic disorder Glutathione-S-transferase (GST) Hepatic disorder Glycogen phosphorylase (GP) Acute myocardial infarction Lactate dehydrogenase (LD) Myocardial infarction Hepatic disorder Hemolysis Carcinoma Lipase (LPS) Acute pancreatitis 5′-Nucleotidase Hepatic disorder Pseudocholinesterase (PChE) Organophosphate poisoning Genetic variants Hepatic disorder Suxamethonium sensitivity Pyruvate kinase (PK) Hemolytic anemia Trypsin (TRY) Acute pancreatitis 18698_ch13_p262-291.indd 270 10/11/12 11:20 AM  CHAPTER 13 n ENZYMES 271 Diagnostic Significance Case Study 13-1 Due to the high concentrations of CK in muscle tis- A 51-year-old, overweight white man visits his f­amily sue, CK levels are frequently elevated in disorders of physician with a symptom of “indigestion” of 5 days’ ­cardiac and skeletal muscle (myocardial infarction [MI], duration. He has also had bouts of sweating, malaise, ­rhabdomyolysis, and muscular dystrophy). The CK level and headache. His blood pressure is 140/105 mm is considered a sensitive indicator of acute myocardial Hg; his family history includes a father with diabetes infarction (AMI) and muscular dystrophy, particularly who died at age 62 of AMI secondary to diabetes the Duchenne type. Extreme elevations of CK occur in mellitus. An electrocardiogram revealed changes Duchenne-type muscular dystrophy, with values reach- from one performed 6 months earlier. The results of ing 50 to 100 times the upper limit of normal (ULN). the patient’s blood work are as follows: Although total CK levels are sensitive indicators of these disorders, they are not entirely specific indicators as CK CK 129 U/L (30–60) elevation is found in various other abnormal cardiac and skeletal muscle conditions. Levels of CK also vary with CK-MB 4% ( LD-2 Elevated CK levels are also occasionally seen in cen- AST 35 U/L (5–30) tral nervous system disorders such as strokes, seizures, nerve degeneration, and central nervous system shock. Questions Damage to the blood–brain barrier must occur to allow enzyme release to the peripheral circulation. 1. Can a diagnosis of AMI be ruled out in this Other pathophysiologic conditions in which elevated patient? CK levels occur are hypothyroidism, malignant hyper- 2. What further cardiac markers should be run on pyrexia, and Reye’s syndrome. Table 13-3 lists the major this patient? disorders associated with abnormal CK levels. Serum CK levels and CK/progesterone ratio have been useful in the 3. Should this patient be admitted to the hospital? diagnosis of ectopic pregnancies.5 Total serum CK levels have also been used as an early diagnostic tool to identify patients with Vibrio vulnificus infections.6 Because enzyme elevation is found in numerous dis- source, diagnostic significance, assay method, source of orders, the separation of total CK into its various isoen- error, and reference range. zyme fractions is considered a more specific indicator of various disorders than total levels. Typically, the clinical Creatine Kinase relevance of CK activity depends more on isoenzyme CK is an enzyme with a molecular weight of approxi- fractionation than on total levels. mately 82,000 Da that is generally associated with ATP CK occurs as a dimer consisting of two subunits that regeneration in contractile or transport systems. Its can be separated readily into three distinct molecular forms. predominant physiologic function occurs in muscle The three isoenzymes have been designated as CK-BB (brain cells, where it is involved in the storage of high-energy type), CK-MB (hybrid type), and CK-MM (muscle type). creatine phosphate. Every contraction cycle of muscle On electrophoretic separation, CK-BB will migrate fastest results in creatine phosphate use, with the production of toward the anode and is therefore called CK-1. CK-BB is ATP. This results in relatively constant levels of muscle followed by CK-MB (CK-2) and, finally, by CK-MM (CK- ATP. The reversible reaction catalyzed by CK is shown 3), exhibiting the slowest mobility (Fig. 13-5). Table 13-3 in Equation 13-4: indicates the tissue localization of the isoenzymes and the CK major conditions associated with elevated levels. Separation Creatine + ATP ∆ Creatine phosphate + ADP of CK isoforms may also be visualized by high-voltage elec- (Eq. 13-4) trophoretic separation. Isoforms occur following cleavage of the carboxyl-terminal amino acid from the M subunit Tissue Source by serum carboxypeptidase N. Three isoforms have been CK is widely distributed in tissue, with highest activities described for CK-MM and two isoforms for CK-MB; the found in skeletal muscle, heart muscle, and brain tissue. clinical significance is not well established. CK is present in much smaller quantities in other tissue The major isoenzyme in the sera of healthy people is sources, including the bladder, placenta, gastrointestinal the MM form. Values for the MB isoenzyme range from tract, thyroid, uterus, kidney, lung, prostate, spleen, undetectable to trace (