| Definition of enzyme activity
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| The activity of an enzyme is obtained by determining the rate of an enzyme-catalyzed reaction under defined conditions. Reaction rate or velocity (v) is generally expressed as the rate of conversion of substrate to product per min, i.e. mol/min. Since the catalytic activity of an enzyme is normally independent of reaction volume, i.e. it is unaffected by dilution, substrate turnover per unit of time under defined conditions (pH, buffer, temperature) is commonly used for defining enzyme-catalyzed reactions.
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| The amount of enzyme activity that catalyzes conversion of 1 mole of substrate into 1 mole of product per second is expressed as the katal (1kat = 1 mol/s). However, the katal is inconvenient for expressing the actual enzyme activity, because it is generally a very small number. The much larger international unit (1 IU = 1μmol/min) is more commonly used as the standard unit of activity.
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| The specific activity of an enzyme, expressed as μmol/min/mg of protein, or IU/mg of protein, indicates the amount of enzyme in a protein sample, and is useful for estimating the purity of an enzyme. The higher the specific activity of an enzyme, i.e. the more units/mg of protein, the higher its purity or homogeneity.
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| Reaction specificity and substrate specificity are determined by the active site structure
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Most enzymes are highly specific for both the type of reaction catalyzed and the nature of the substrate(s). Reaction specificity, i.e. the reaction that the enzyme catalyzes, is determined chemically by the amino acid residues in the catalytic center of the enzyme. In general, the active site of the enzyme is composed of the substrate binding site and the catalytic site. Substrate specificity is determined by the size, structure, charges, polarity, and hydrophobicity of the substrate binding site. This is because the substrate must bind in the active site as the first step in the reaction, setting the stage for catalysis. Highly specific enzymes such as catalase and urease, which degrade H2O2 and urea , respectively, catalyze only one type of reaction, but some enzymes have broader substrate specificity. The serine proteases are a typical example of such a group of enzymes. These are a family of closely related enzymes, such as the pancreatic enzymes, chymotrypsin, trypsin, and elastase, which contain a reactive serine residue in the catalytic site. They catalyze the hydrolysis of peptide bonds on the carboxyl side of a limited range of amino acids in protein. Although they have similar structures and catalytic mechanisms, their substrate specificities are quite different because of structural features of the substrate binding site (Fig. 5.2).
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| Isozymes are enzymes that catalyze the same reaction, but differ in their primary structure and/or subunit composition. Levels of some tissue-specific enzymes and isozymes are measured in serum for diagnostic purposes (Fig. 5.3 and Table 5.1).
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Table 5-1.
Some enzymes used for clinical diagnosis of disease. |
| Body_ID: None |
| Some enzymes used for clinical diagnosis of disease |
| Body_ID: T005001.50 |
| Enzyme | Tissue source(s) | Diagnostic use |
| Body_ID: T005001.100 |
| AST | heart, skeletal muscle, liver, brain | liver disease |
| Body_ID: T005001.150 |
| ALT | liver | liver disease, e.g. hepatitis (ALT > AST) |
| Body_ID: T005001.200 |
| amylase | pancreas, salivary gland | acute pancreatitis, biliary obstruction |
| Body_ID: T005001.250 |
| CK | skeletal muscle, heart, brain | muscular dystrophy, myocardial infarction |
| Body_ID: T005001.300 |
| GGT | liver | hepatitis, alcohol excess |
| Body_ID: T005001.350 |
| LDH | heart, liver erythrocytes | lymphoma, hepatitis |
| Body_ID: T005001.400 |
| lipase | pancreas | acute pancreatitis, biliary obstruction |
| Body_ID: T005001.450 |
| alkaline phosphatase | osteoblast | bone disease, bone tumors |
| Body_ID: T005001.500 |
| acid phosphatase | prostate | prostate cancer |
| Body_ID: T005001.550 |
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| Body_ID: T005001.600 |
AST = aspartate amino transferase; ALT = alanine amino transferase; CK = creatine phosphokinase; GGT = gamma glutamyl transferase; LDH = lactate dehydrogenase.
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| page 52 |  | | page 53 |
| Figure 5.2 Characteristics of the substrate-binding sites in the serine proteases chymotrypsin, trypsin, and elastase. In chymotrypsin a hydrophobic pocket binds aromatic amino acid residues such as phenylalanine (Phe). In trypsin, the negative charge of the aspartate residue in the substrate binding site promotes cleavage to the carboxyl side of positively charged lysine (Lys) and arginine (Arg) residues. In elastase, side chains of valine and threonine block the substrate binding site and permit binding of amino acids with small or no side chains, such as glycine (Gly). |
| Figure 5.3 Densitometric patterns of the LDH isozymes in serum of patients diagnosed with myocardial infarction or acute hepatitis. Isozymes, differing slightly in charge, are separated by electrophoresis on cellulose acetate, visualized using a chromogenic substrate, and quantified by densitometry. Total serum LDH activity is also increased in these patients. Since hemolysis releases LDH from red blood cells and affects diagnosis, blood samples should be treated with care. The LDH measurements for the diagnosis of myocardial infarction have now been superseded by plasma troponin levels. |
| page 53 | | | page 54 |
| TISSUE SPECIFICITY OF LDH ISOZYMES |
| A 56-year-old female was admitted to an intensive care unit. The patient had suffered from a slight fever for 1 week, and had some chest pain, and difficulty breathing for the past 24 h. No abnormality was found on chest X-ray or by electrocardiography. However, a blood test showed white blood cells 12100/mm3 (normal: 4000 - 9000/mm3), red blood cells 240 × 104/mm3 (normal: 380 - 500 × 104/mm3), hemoglobin 8.6 g/dL (normal: 11.8 - 16.0 g/dL), lactate dehydrogenase (LDH) 1400 IU/L (normal: 200 - 400 IU/L). Levels of other enzymes were normal. Based on the blood tests, the LDH isozyme profile and other data, the patient was eventually diagnosed with malignant lymphoma. |
| Comment. LDH is a tetrameric enzyme composed of two different 35 kDa subunits. The heart contains mainly the H type, and skeletal muscle and the liver the M type subunit, which are encoded by different genes. Five types of tetrameric isozymes can be formed from these subunits: H4 (LDH1), H3M1 (LDH2), H2M2 (LDH3), H1M3 (LDH4), and M4 (LDH5). Since isozyme distributions differ among tissues, it is possible to diagnose tissue damage by assaying total LDH activity and then by isozyme profiling (Fig 5.3). |
| For hematological reference values, see Table 4.2. |
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| A systematic classification is required to organize the different enzymes that catalyze the many thousands of reactions that take place in our body. All enzymes are assigned a four-digit enzyme classification (EC) number. The first digit indicates membership of one of the six major classes of enzymes shown in Table 5.2. The next two digits indicate substrate subclasses and sub-subclasses. For example, the transfer of reducing equivalents from one redox system to another is catalyzed by the oxidoreductases (Class 1). The transfer of other functional groups from one substrate to another is catalyzed by the transferases (Class 2). The hydrolases (Class 3) catalyze group transfer, but the acceptor molecule is exclusively a water molecule. Reactions involving the addition or removal of H2O, NH3, or CO2 are catalyzed by lyases (Class 4),
also called synthases. Isomerases (Class 5) catalyze isomerization reactions by rearranging atoms within a molecule, and thus do not affect the composition of the substrate. Ligases, also called synthetases (Class 6), use ATP to catalyze energy-dependent synthetic reactions.
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