Enzymes relative specificity

AMP aminohydrolase, an enzyme relatively specific for AMP, has been observed in reptiles (44), erythrocytes (38), snail (45), unfertilized fish eggs (46), invertebrates (47), a variety of mammalian tissues (20), and a particulate fraction of pea seeds (48). Evidence suggests that the frog muscle AMP aminohydrolase is located within or just beneath the sarcolemma (49). The rabbit skeletal and heart muscle enzymes were found in the cytoplasm and mitochondria (20, Jfi, 50, 51), while the enzyme of kidneys and gills of freshwater fish was located in the cytoplasmic fraction (52). The enzyme occurs in most areas of the rat (53) and rabbit brain (54). The nonspecific enzyme from several microbial sources deaminates adenosine triphosphate (ATP) and adenosine diphosphate (ADP) as well as AMP (see Section V). 

Effect of an ilv S Mutation on Isoleucine Valine Biosynthetic Enzymes Relative Specific Activity"... 

In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. Cysteine residues in proteins, for example, react with one another to form disulfide species and also react with a number of reagents, including maleimides (typically A ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively... 

Disulfoton and its breakdown products can be measured in the blood, urine, feces, liver, kidney, or body fat of exposed people. In cases of occupational or accidental exposure to disulfoton, the breakdown products are often measured in the urine. The breakdown products are relatively specific for disulfoton and a few other similar organophosphate pesticides and can be detected in urine for up to one week after people were last exposed. Because disulfoton inhibits cholinesterase in blood and in blood cells, inhibition of this enzyme activity may also suggest exposure to disulfoton. Cholinesterase activity in blood and in blood cells may remain inhibited for as long as 1-2 weeks after the last exposure. Because other organophosphate pesticides also inhibit cholinesterase activity in blood and blood cells, this test is not specific for disulfoton. The measurement of cholinesterase in blood and blood cells and the amount of disulfoton breakdown products in the urine cannot always predict how much disulfoton you were exposed to. Your doctor can send samples of your blood or urine to special laboratories that perform these tests. Chapters 2 and 6 provide more information about medical tests. 

Figure 2. Distribution of marker enzymes and DEHP-metabolizing enzymes in trout liver homogenate fractions. DEHP esterase and DEHP oxidase were each measured by 1-hr incubations of 0.010 ftmol of UC-DEHP in a total volume of 2 mL. Fraction (A), 2,000 g pellet (B), 10,000 g pellet (C), 100,000 g pellet and (D), 100,000 g supernatant. Relative Specific Activity = percent of total activity/ percent of total protein (14).

The presence of calcium in horseradish peroxidase was demonstrated originally by Haschke and Friedhoff, working with the C and A (imspec-ified, but likely to have been predominantly A2) isoenzymes (209). HRP C and HRP A contain 2.0 0.13 and 1.4 0.19 moles calcium per mole enzyme, respectively, as determined by atomic absorption spectroscopy. Incubation in 6 M guanidinium hydrochloride and 10 mM EDTA for 4 hours at neutral pH and room temperature gave calcium-depleted enzymes with specific activities decreased by 40% and 15%, respectively. The thermal stability of calcium-depleted HRP C was also reduced compared to native enzyme. Reconstitution was successful only with calcium-depleted HRP C (209). It remains to be established whether this reflects true structural differences between the calcium binding sites of the two isoenz5unes, or is a consequence of the relatively harsh... 

The value and potential usefulness of a new enzyme depends on its properties and the extent to which it has been characterized. The initial characterization of an enzyme often involves the determination of its pH optimum, stability, gross physical properties, and substrates. The enzymes of L. edodes, typically show pH optima between 3.5 and 5.0, maximal activity at 50 to 60"C, little activity loss until over 70"C, and high relative specific activities (9,14). Below we will highlight some of the other characteristics determined for the major ligninase, p-(l,4)-D-xylanase, and a-(l,3)-L-arabinosidase purified from wood-grown cultures of L. edodes. 

The proteolytic enzymes are classified into endopeptidases and exopeptidases, according to their site of attack in the substrate molecule. The endopeptidases or proteinases cleave peptide bonds inside peptide chains. They recognize and bind to short sections of the substrate s sequence, and then hydrolyze bonds between particular amino acid residues in a relatively specific way (see p. 94). The proteinases are classified according to their reaction mechanism. In serine proteinases, for example (see C), a serine residue in the enzyme is important for catalysis, while in cysteine proteinases, it is a cysteine residue, and so on. 

Similar explanations, supported by convincing evidence, have been proposed for the high catalytic efficiency and specificity of enzymes (particularly hydrolytic enzymes) relative to ordinary catalysts (Laidler, 23, Wilson, 65, 66). 

Conversely, if [S] < C Km, (Eq. (2.42)) reduces to v = (k2/Km)[E]o[S]. This means that the active sites on the enzyme are effectively unoccupied. The ratio k2/Km is also known as the enzyme s specificity constant, a measure of the enzyme s affinity for different substrates. Thus, if the same enzyme can catalyze the reaction of two substrates, S and S, the relative rates of these two reactions are compared using (k2/Km)s (k2/Km)s-. Because the specificity constant reflects both affinity and catalytic ability, it is also used for comparing different enzymes. 

Undoubtedly, mimetic models of enzymes must conform to definite physicochemical features of the target enzyme. The specific feature of any biomimic is its relatively small size and simpler structure. 

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase (see Topic C5). These three enzymes belong to a family of enzymes called the serine proteases - serine because they have a serine residue in the active site that is critically involved in catalysis and proteases because they catalyze the hydrolysis of peptide bonds in proteins. The three enzymes cleave peptide bonds in protein substrates on the carboxyl side of certain amino acid residues. 

If we are quite certain that ACE immobilized at the surface of the endotheliaf celfs is physiologicaliy more important than is the circulating enzyme (197), we do not know yet what is the relative importance of its bland C-terminal active sites. We do not know how they possibly affect the activity of each other. We do not know the physiological role of the N-acetyl SDKP, a relatively specific substrate of the N-terminal active site. We may still discover other natural substrates for endothelial and epithelial ACE. We do not fully understand the basis of differences in the various daily doses of ACE inhibitors as usually prescribed, from trandolapril 2 mg/day to lisinopril 80 mg/day, and the consequences of dose choices. 

It is clear that the formation of an enzyme-substrate complex is required for both the catalytic action of an enzyme and its substrate specificity. Substrates are bound to a relatively small region of the enzyme called the active or catalytic site, which contains specific residues directly responsible for catalytic action. The active site is three-dimensional and is formed by groups from different areas of the linear polypeptide sequence. Activity therefore depends on the integrity of the three-dimensional structure of the enzyme. The specificity of binding depends upon a complementary relationship between the enzyme active site and the substrate. 

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