Enzyme and rate analysis

Background

Enzymes are biological catalysts that can be used in the diagnosis and monitoring of disease and their remarkable properties make them sensitive indicators of pathologic change.

Metabolism can be regarded as an integrated series of enzymatic reactions and some diseases as a derangement of the physiologic pattern of metabolism. Many enzymes exist in multiple forms, and differences in their properties help in differentiating them and understanding organ-specific pathophysiology. Genetically determined variations in enzyme structure among individuals are used to account for such characteristics as differences in sensitivity to drugs and differences in metabolism that manifest as hereditary metabolic diseases.

Content

The properties and mechanism of action of enzymes that influence the specificity and sensitivity of these proteins and enables them to be used in disease management are described. In addition, the existence of multiple forms of some enzymes provides opportunities to increase the diagnostic specificity and sensitivity of enzyme assays in body fluids. The principles of enzyme kinetics are described, as well as how these properties are affected by activators and inhibitors. Kinetic properties are used to develop optimal conditions for measuring catalytic concentrations of enzymes and standardization approaches in order to achieve comparable results in clinical samples have been proposed. Finally, the use of enzymes as biological reagents in the measurement of metabolites and as indicator reactions in many immunoassays is mentioned.

Enzymes are proteins with catalytic properties, and clinical enzymology is the application of the science of enzymes to the diagnosis and monitoring of disease. The principles of clinical enzymology will be introduced and discussed in this chapter, as will information on how enzymes are measured and how they are used as analytical reagents in various types of rate analysis. Individual topics include basic principles, enzyme kinetics, analytical enzymology, and rate analyses.

Enzyme nomenclature

Historically, individual enzymes were identified using the name of the substrate or group on which they act and then adding the suffix - ase. For example, the enzyme hydrolyzing urea was ure ase. Later, the type of reaction involved was identified, as in carbonic anhydrase, d -amino acid oxidase, and succinate dehydrogenase. In addition, some enzymes were given empirical names such as trypsin, ptyalin, pepsin, and emulsin.

Because this combination of trivial common names and semisystematic names was found to be inadequate, in 1955 the International Union of Biochemistry (IUB) appointed an Enzyme Commission (EC) to study the problem of enzyme nomenclature. Its subsequent recommendations, with periodic updating, provide a rational and practical basis for identifying all enzymes now known and enzymes that will be discovered in the future.

With the IUB system, a systematic and trivial name is provided for each enzyme. The systematic name describes the nature of the reaction catalyzed and is associated with a unique numeric code. The trivial or practical name, which may be identical to the systematic name but is often a simplification of it, is suitable for everyday use. The unique numeric designation for each enzyme consists of four numbers, separated by periods (e.g., 2.2.8.11), and is prefixed by the letters EC, denoting Enzyme Commission. The first number defines the class to which the enzyme belongs. All enzymes are assigned to one of six classes, characterized by the type of reaction they catalyze: (1) oxidoreductases, (2) transferases, (3) hydrolases, (4) lyases, (5) isomerases, and (6) ligases. The next two numbers indicate the subclass and the sub-subclass to which the enzyme is assigned. For example, these may differentiate the amino-transferring subclass from the phosphate-transferring category, or the ethanol acceptor sub-subclass from that accepting acyl groups. The last number is the specific serial number given to each enzyme within its sub-subclass.

To illustrate how this system is used to name an enzyme, consider the enzyme creatine kinase (CK), which catalyzes the following reaction:

Its EC system number is

Table 25.1 lists selected enzymes of clinical interest, identified by trivial, abbreviated, and systematic names and by their code numbers.

Although not recommended by the EC, it is a common and convenient practice to use capital letter abbreviations for the names of certain enzymes, such as ALT for alanine aminotransferase, AST for aspartate aminotransferase, LDH for lactate dehydrogenase, and CK for creatine kinase (see Table 25.1 ).

Enzymes as proteins

Enzymes are proteins that possess the primary, secondary, and tertiary structural characteristics of proteins (see Chapter 31 ), and most also exhibit quaternary structure. The primary structure, the linear sequence of amino acids linked through their α-carboxyl and α-amino groups by peptide bonds, is specific for each type of enzyme molecule. Each polypeptide chain is coiled into three-dimensional (3D) secondary and tertiary structures. Secondary structure refers to the conformation of limited segments of the polypeptide chain, namely α-helices, β-pleated sheets, random coils, and β-turns. The arrangement of secondary structural elements and amino acid side chain interactions that define the 3D structure of the folded protein is referred to as its tertiary structure. In many cases, biological activity, such as the catalytic activity of enzymes, requires two or more folded polypeptide chains (subunits) to associate to form a functional molecule. The arrangement of these subunits defines the quaternary structure. The subunits may be copies of the same polypeptide chain (e.g., the MM isoenzyme of CK, the H ₄ isoenzyme of LDH), or they may represent distinct polypeptides (e.g., the MB isoenzyme of CK).

Considerable homologies of sequence are found between enzymes that appear to share a common evolutionary origin, such as the proteases trypsin and chymotrypsin, and similarities of sequence are even more marked among the members of a family of isoenzymes. The amino acid sequence in the immediate neighborhood of the active center of the enzyme (discussed later) is often similar in enzymes with related function (e.g., the serine proteases are so called because they all have this amino acid in the active center).

Enzyme molecules differ in the proportion of secondary structures—such as α-helices—they contain, although no enzyme molecule studied thus far approaches the large proportion of α-helices found in myoglobin and hemoglobin. The tertiary structures of different types of enzyme molecules are as individually characteristic as their primary structures; nevertheless, some common features exist. Enzyme molecules are roughly globular in overall shape, with a preponderance of polar amino acid side chains on the outside of the molecule and nonpolar side chains in the interior. The ionizable residues in contact with the surrounding medium are responsible for many of the properties of the enzyme molecules in solution, such as their migration in an electric field and their solubility. Covalent disulfide bridges may link different parts of the polypeptide chains in some enzyme molecules, but the 3D structure is mainly stabilized by the large number of hydrophobic interactions formed between the nonpolar side chains in the interior of the molecule.

The application of physical methods, such as x-ray crystallography and multidimensional nuclear magnetic resonance, has provided structural insights on which enzyme mechanisms have been built. Furthermore, the tools of molecular biology, such as molecular cloning, have enabled the purification and characterization of enzymes that previously were available only in minute amounts. Molecular biology also enables the manipulation of the amino acid sequence of enzymes, and site-directed mutagenesis (substituting one amino acid residue for another) and deletion mutagenesis (eliminating sections of the primary structure) have enabled the identification of chemical groups that participate in ligand binding and specific chemical steps during catalysis. There are a number of free online databases describing various properties of enzymes, including structure and reaction mechanisms.

The biological activity of a protein molecule depends generally on the integrity of its structure, and any disruption of its structure is accompanied by a loss of activity, a process known as denaturation. If the process of denaturation is minimal, it may be reversed with the recovery of enzyme activity on removal of the denaturing agent. However, prolonged or severe denaturing conditions result in an irreversible loss of activity. Denaturing conditions include increased temperatures, extremes of pH, and chemical addition. Heat inactivation of most enzymes takes place at an appreciable rate at room temperature and in most cases becomes almost instantaneous above 60 °C. The polymerases are an exception and retain activity at temperatures as high as 90 °C—a property that has been made use of in the polymerase chain reaction (see Chapter 62 ). Low temperatures are used to preserve enzyme activity, especially in aqueous solutions, such as serum (see Chapter 32 ). Extremes of pH also cause unfolding of enzyme molecular structures and, except for a few exceptions, should be avoided when enzyme samples are preserved. Addition of chemicals, such as urea and detergents, disrupts hydrogen bonds and hydrophobic interactions so that exposure of enzymes to strong solutions of these reagents results in inactivation.

Specificity and the active center

With the exception of enzymes such as proteases, nucleases, and amylases, which act on macromolecular substrates, enzyme molecules are considerably larger than the molecules of their substrates. Consideration of the structure of an enzyme’s active site and its relationship to the structures of the enzyme’s substrate(s) in its ground and transition states is necessary to understand the rate enhancement and specificity of the chemical reactions performed by an enzyme. The active site of an enzyme will vary among enzymes but in general :

• 1.

  The active site of an enzyme is relatively small compared with the total size of the enzyme molecule and may involve less than 5% of the total amino acids in the molecule.

• 2.

  The active sites of enzymes are 3D structures that are formed as a result of the overall tertiary structure of the protein. This results in the amino acids and cofactors in the active site of an enzyme being spatially structured in an exact, 3D relationship with respect to one another and reflects the structure of the substrate molecule.

• 3.

  Typically, the attraction between the molecules of the enzyme and its substrate molecules is noncovalent binding. Physical forces used in this type of binding include hydrogen bonding, electrostatic and hydrophobic interactions, and van der Waals interactions.

• 4.

  Active sites of enzymes typically occur in clefts and crevices in the protein.

The specificity of substrate binding is a function of the exact spatial arrangement of atoms in the enzyme active site that complements the structure of the substrate molecule.

Isoenzymes and other multiple forms of enzymes

The IUB recommends that isoenzyme or isozyme should apply only to those multiple forms of enzymes arising from genetically determined differences in primary structure and not to those derived by modification of the same primary sequence. The term multiple forms of the enzyme should be used as a broad term covering all proteins catalyzing the same reaction and occurring naturally in a single species, for example, to an enzyme that has undergone post-translational modification.

Isoenzymes may occur within a single organ or even within a single type of cell. The forms can be distinguished on the basis of differences in various physical properties, such as electrophoretic mobility and resistance to chemical or thermal inactivation. They often have significant quantifiable differences in catalytic properties, but all forms of a particular enzyme retain the ability to catalyze its characteristic reaction.

The existence of multiple forms of enzymes in human tissue has important implications in the study of human disease. The presence in different organs of isoenzymes with distinctive properties helps in understanding organ-specific patterns of metabolism, but genetically determined variations in enzyme structure among individuals account for such characteristics as differences in sensitivity to drugs and differences in metabolism, which manifest themselves as hereditary metabolic diseases. For diagnostic enzymology, the existence of multiple forms of enzymes, whether the result of genetic or nongenetic causes, provides opportunities to increase the diagnostic specificity and sensitivity of enzyme assays in body fluid samples.

Similar to other proteins, enzymes usually elicit the production of antibodies when they are injected into animals of a species other than those in which they originate. Even small structural differences among closely similar molecules, such as the members of a family of isoenzymes, are often sufficient to render them antigenically distinct, allowing antibodies to be produced specific to a single type of molecule. The availability of enzyme-specific antisera opens up a wide range of methods in enzyme analysis, some of which—such as immunoassay—do not depend on the catalytic activity of the enzyme molecules. The availability of immunochemical methods has been particularly important in the analysis of isoenzyme mixtures. Many commercial immunoassays now use monoclonal antibodies to increase analytical selectivity.

Genetic origins of enzyme variants

True isoenzymes result from the existence of more than one gene locus coding for the structure of the enzyme protein. Many human enzymes (more than one-third) are known to be determined by more than one structural gene locus. Genes at the different loci have undergone differential modifications during the evolution, so that the enzyme proteins coded by them no longer have identical structures, although they are recognizably similar; in other words, they are isoenzymes.

The multiple genes that determine a particular group of isoenzymes are not necessarily closely linked on one chromosome. For example, the structural genes that code for human salivary and pancreatic amylases both are located on chromosome 1, whereas the genes that code for cytoplasmic and mitochondrial malate dehydrogenase are carried on chromosomes 2 and 7, respectively. Among the enzymes of clinical importance that exist as isoenzymes because of the presence of multiple-gene loci are LDH, CK, α-amylase, and some forms of alkaline phosphatase (ALP).

An enzyme can exist in molecular forms that differ from one individual to another because of the existence of alternative alleles that are inherited according to mendelian laws. These give rise to gene products with the same function and isoenzymes that result from the existence of allelic genes that are termed allozymes. The proportion of human gene loci subject to allelic variation is considerable, and the probability that individual human beings will differ to some degree in their isoenzyme patterns is correspondingly high.

The number of allelic variants and the frequency with which particular variants occur within the population vary considerably across enzymes. For example, mutations at either of the two principal loci that determine human LDH are extremely rare, but a high incidence of mutant alleles occurs at the single locus that determines the structure of placental ALP. More than 400 mutations in the glucose-6-phosphate dehydrogenase gene have now been identified on the X chromosome (up-to-date genetic information on this and other enzymes can be obtained from the Online Mendelian Inheritance in Man [OMIM] database ). When isoenzymes, because of variation at a single locus, occur with appreciable frequency in a human population, the population is said to be polymorphic with respect to the isoenzymes in question.

Another category of multiple molecular forms can arise when enzymes are oligomeric, consisting of molecules made up of subunits. The association of different types of subunits in various combinations gives rise to a range of active enzyme molecules. When the subunits are derived from different structural genes—multiple loci or multiple alleles—the hybrid molecules so formed are called hybrid isoenzymes. The ability to form hybrid isoenzymes is evidence of considerable structural similarities among the different subunits. Hybrid isoenzymes can be formed in vitro, but they are also formed in vivo in cells in which different types of constituent subunits are present in the same subcellular compartment.

The number of different hybrid isoenzymes that can be formed from two nonidentical protomers depends on the number of subunits in the complete enzyme molecule. For a dimeric enzyme, one mixed dimer (hybrid isoenzyme) can be formed. If the enzyme is a tetramer, three heteropolymeric isoenzymes may be formed ( Fig. 25.1 ). Examples of hybrid isoenzymes include the mixed MB dimer of CK and the three hybrid isoenzymes, LDH-2, LDH-3, and LDH-4, of LDH.

Nongenetic causes of multiple forms of enzymes

Post-translational modifications of enzyme molecules give rise to multiple forms known as isoforms ( Fig. 25.2 ).

Modifications of residues in the polypeptide chains of enzyme molecules take place in living cells to yield multiple forms. For example, removal of amide groups accounts for some of the heterogeneity of amylase and carbonic anhydrase (each of these enzymes also exists as a true isoenzyme). Modification also can take place as a result of extraction procedures. Many erythrocyte enzymes, including adenosine deaminase, acid phosphatase, and some forms of phosphoglucomutase, contain sulfhydryl groups that are susceptible to oxidation. In hemolysates, oxidation may be brought about by the action of oxidized glutathione, although in intact cells, this compound is present in its reduced form. Thus variant enzyme molecules with altered molecular charge may be generated.

Serum isoforms of CK are formed as part of the normal clearance process of the enzyme. Human myocardial and skeletal muscle tissues have the CK-MM and CK-MB isoenzymes, which are modified on release into the circulation. This modification is due to sequential removal of the C-terminal amino acid, lysine, by the action of carboxypeptidase.

Modifications affecting nonprotein components of enzyme molecules also may lead to molecular heterogeneity. Many enzymes are glycoproteins, and variations in carbohydrate side chains are a common cause of nonhomogeneity of preparations of these enzymes. Some carbohydrate moieties, notably N -acetylneuraminic acid (sialic acid), are strongly ionized and consequently have a profound effect on some properties of enzyme molecules. For example, removal of terminal sialic acid groups from human liver and/or bone ALP with neuraminidase greatly reduces the electrophoretic heterogeneity of the enzyme.

Aggregation of enzyme molecules with each other or with nonenzymatic proteins may give rise to multiple forms that can be separated by techniques that depend on differences in molecular size. For example, four catalytically active cholinesterase components with molecular weights ranging from approximately 80,000 to 340,000 are found in most sera, with the heaviest component, carbon (C ₄ ), contributing most of the enzyme activity. Other enzyme forms are also occasionally present, but it appears that the principal serum cholinesterase fractions can be attributed to different states of aggregation of a single monomer.

A specific form of interaction between enzymatic and nonenzymatic proteins, such as immunoglobulins, results in the formation of an enzyme-protein complex (“macrocomplex”). Since the identification of macroamylase, the first such enzyme–immunoglobulin complex to be identified, similar complexes have been observed involving LDH, CK, ALP, and other enzymes.

A single polypeptide chain in theory exists in an infinite number of different conformations. However, one specific conformation generally appears to be the most stable for any given sequence of amino acids, and this conformation is assumed by the chain as it is synthesized within the cell. Thus the primary structure of the polypeptide chain also determines its 3D secondary and tertiary structures. It is conceivable that in some cases, several alternative conformations (conformers) of a single chain that are almost equally stable may be present, and therefore these alternative forms may coexist. However, no multiple-enzyme forms have been shown unequivocally to be due to conformational isomerism.

Enzymes as catalysts

A catalyst is a substance that modifies and increases the rate of a chemical reaction without being consumed or permanently altered. Enzymes are protein catalysts of biological origin. Metabolism is a coordinated series of chemical reactions that occur within a living cell to provide energy and accomplish biosynthesis. The process can be regarded as an integrated series of enzymatic reactions and some diseases as a derangement of the normal pattern of metabolism. Apart from these fundamental considerations, it is the remarkable properties of enzymes that make them such sensitive indicators of pathologic change.

Because of their catalytic activity, a given number of enzyme molecules convert an enormous number of substrate molecules to products within a short time. This property is used to measure increased amounts of enzymes in the bloodstream, although the amount of enzyme protein released from damaged cells is small compared with the total quantity of nonenzymatic proteins in blood. Thus a change in the quantity of a particular enzyme is recognized by its characteristic effect on a given chemical reaction.

Units for expressing enzyme activity

When enzymes are measured by their catalytic activities, the results of such determinations are expressed in terms of the concentration of the number of activity units present in a convenient volume or mass of specimen. The unit of activity is the measure of the rate at which the reaction proceeds (e.g., the quantity of substrate consumed, or product formed in a chosen unit of time). In clinical enzymology, the activity of an enzyme is generally reported in terms of unit of volume, such as activity per liter of serum, or per 1.0 mL of packed erythrocytes. Because the rate of the reaction depends on experimental parameters, such as pH, type of buffer, temperature, nature of substrate, ionic strength, concentration of activators, and other variables, these parameters must be specified in the definition of the unit.

To standardize how enzyme activities are expressed, the EC proposed that the unit of enzyme activity should be defined as the quantity of enzyme that catalyzes the reaction of 1 μmol of substrate per minute and that this unit should be termed the international unit (U). Catalytic concentration is to be expressed in terms of units per liter (U/L) or kilounits (kU/L), whichever gives the more convenient numeric value. In this chapter, the symbol U is used to denote the international unit. In those instances in which there is some uncertainty about the exact nature of the substrate, or when difficulty is encountered in calculating the number of micromoles reacting (as with macromolecules such as starch, protein, and complex lipids), the unit is to be expressed in terms of the chemical group or residue measured in the following reaction (e.g., glucose units, amino acid units formed).

The International System of Units (SI)-derived unit for catalytic activity is the katal, defined as moles converted per second. The name katal had been used for this unit for decades but did not become an official SI-derived unit until 1999. Both the International Union of Pure and Applied Chemistry and the IUB recommend that enzyme activity be expressed in moles per second and that the enzyme concentration be expressed in terms of katals per liter (kat/L). Thus 1 U = 10 ⁻⁶ mol/60 s = 16.7 × 10 ⁻⁹ mol/s, or 1.0 nkat/L = 0.06 U/L.

The enzyme-substrate complex

Enzymes act through the formation of an enzyme–substrate (ES) complex, in which a molecule of substrate is bound to the active center of the enzyme molecule. The binding process transforms the substrate molecule to its activated state. The energy required for this transformation is provided by the free energy of binding of S to E. Therefore activation takes place without the addition of external energy, so that the energy barrier to the reaction is lowered and the breakdown to products is accelerated ( Fig. 25.3 ). The ES complex breaks down to give the reaction products (P) and free enzyme (E):

All reactions catalyzed by enzymes are in theory reversible. However, in practice, the reaction is usually found to be more rapid in one direction than in the other, so that an equilibrium is reached in which the product of the forward or the backward reaction predominates, sometimes so markedly that the reaction is virtually irreversible.

If the product of the reaction in one direction is removed as it is formed (e.g., because it is the substrate of a second enzyme present in the reaction mixture), the equilibrium of the first enzymatic process will be displaced so that the reaction will proceed to completion in that direction. Reaction sequences in which the product of one enzyme-catalyzed reaction becomes the substrate of the next enzyme and so on, often through many stages, are characteristic of biological processes. In the laboratory also, several enzymatic reactions may be linked together to provide a means of measuring the activity of the first enzyme or the concentration of the initial substrate in the chain. For example, the activity of CK is usually measured by a series of linked reactions, and the concentration of glucose is determined by consecutive reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase.

When a secondary enzyme-catalyzed reaction, known as an indicator reaction, is used to determine the activity of a different enzyme, the primary reaction catalyzed by the enzyme to be determined must be the rate-limiting step. Reaction conditions must ensure that the rate of reaction catalyzed by the indicator enzyme is directly proportional to the rate of product formation in the first reaction.