Enzymes absolute specificity

Even though these enzymes have no absolute specificity, many of them show a preference for a particular side chain before the scissile bond as seen from the amino end of the polypeptide chain. The preference of chymotrypsin to cleave after large aromatic side chains and of trypsin to cleave after Lys or Arg side chains is exploited when these enzymes are used to produce peptides suitable for amino acid sequence determination and fingerprinting. In each case, the preferred side chain is oriented so as to fit into a pocket of the enzyme called the specificity pocket. 

T"he extraordinary ability of an enzyme to catalyze only one particular reaction is a quality known as specificity (Chapter 14). Specificity means an enzyme acts only on a specific substance, its substrate, invariably transforming it into a specific product. That is, an enzyme binds only certain compounds, and then, only a specific reaction ensues. Some enzymes show absolute specificity, catalyzing the transformation of only one specific substrate to yield a unique product. Other enzymes carry out a particular reaction but act on a class of compounds. For example, hexokinase (ATP hexose-6-phosphotransferase) will carry out the ATP-dependent phosphorylation of a number of hexoses at the 6-posi-tion, including glucose. 

The absolute quantity of an enzyme reflects the net balance between enzyme synthesis and enzyme degradation, where 4 and represent the rate constants for the overall processes of synthesis and degradation, respectively. Changes in both the 4 and of specific enzymes occur in human subjects. 

Many enzymes have absolute specificity for a substrate and will not attack the molecules with common structural features. The enzyme aspartase, found in many plants and bacteria, is such an enzyme [57], It catalyzes the formation of L-aspartate by reversible addition of ammonia to the double bond of fumaric acid. Aspartase, however, does not take part in the addition of ammonia to any other unsaturated acid requiring specific optical and geometrical characteristics. At the other end of the spectrum are enzymes which do not have specificity for a given substrate and act on many molecules with similar structural characteristics. A good example is the enzyme chymotrypsin, which catalyzes hydrolysis of many different peptides or polypeptides as well as amides and esters. 

Absolute specificity, in which the enzyme catalyzes a single reaction. 

The enzyme has been found in Leuconostoc mesenteroides66 and Pseudomonas saccharophila.6 In a series of outstanding researches Doudoroff and Hassid with associates investigated the specificity requirements of sucrose phosphorylase from P. saccharophila. It was found that the enzyme exhibits an absolute specificity for the D-glucose moiety of its... 

One area of research interest has been the metal ion specificity of the MnSOD and FeSOD molecules. The tertiary structures of these molecules are very similar and the ligands coordinated to the metal ions are identical. Many organisms contain both forms of the enzyme and each form has an absolute specificity for its metal ion, the enzyme is completely inactive if the wrong metal ion is present. Cambialistic enzymes that occur in some organisms are active with either metal ion present in the active site. Comparisons of the structures of the MnSOD, FeSOD, and the cambialistic enzymes have not revealed any single obvious structural differences that could explain this phenomenon. " ... 

This enzyme catalyzes the transamination of a wide spectrum of a-amino acids and a-keto (or 2-oxo) acids, demonstrating absolute specificity for their D-isomers. The most likely physiologic role is to provide D-amino acids for peptidoglycan synthesis in bacterial cell wall formation. 

However, apart from absolute specificity, foreign compounds may also be substrates for enzymes involved in endogenous pathways, often with toxicological consequences. Thus, for example, with VPA (see above), fluoroacetate, and galactosamine (see chap. 7) involvement in endogenous metabolic pathways is a crucial aspect of... 

In the third step, 1, -/3-hydroxyacyl-CoA is dehydrogenated to form /3-ketoacyl-CoA, by the action of /3-hydroxyacyl-CoA dehydrogenase NAD+ is the electron acceptor. This enzyme is absolutely specific for the l stereoisomer of hydroxyacyl-CoA The NADH formed in the reaction donates its electrons to NADH dehydrogenase, an electron carrier of the respiratory chain, and ATP is formed from ADP as the electrons pass to 02. The reaction catalyzed by /3-hydroxyacyl-CoA dehydrogenase is closely analogous to the malate dehydrogenase reaction of the citric acid cycle (p. XXX). 

Ochoas group reported that in their malic enzyme, Co2+ could replace the Mn2+ requirement, but that Mg2+ was considerably less effective. Macrae (31) reported that malic enzyme from cauliflower bud mitochondria has an absolute requirement for either Mn2+, Co2+, or Mg2+. Morenzoni (6) has shown that the NADH producing activity of Leuconostoc oenos exhibited an absolute specificity for Mn2+ Co2+ and Mg2+ could not substitute, nor could Fe3+, Zn2+, or Cu+. Furthermore, Cu2+ inhibits this activity as well as the malo-lactic acivity. 

A second enzyme (of mass 100 kDa) is needed for activation of phenylalanine. It is apparently the activated phenylalanine (which at some point in the process is isomerized from l- to D-phenylalanine) that initiates polymer formation in a manner analogous to that of fatty acid elongation (Fig. 17-12). Initiation occurs when the amino group of the activated phenylalanine (on the second enzyme) attacks the acyl group of the aminoacyl thioester by which the activated proline is held. Next, the freed imino group of proline attacks the activated valine, etc., to form the pentapeptide. Then two pentapeptides are joined and cyclized to give the antibiotic. The sequence is absolutely specific, and it is remarkable that this relatively small enzyme system is able to carry out each step in the proper sequence. Many other peptide antibiotics, such as the bacitracins, tyrocidines,215 and enniatins, are synthesized in a similar way,213 216 217 as are depsipeptides and the immunosuppresant cyclosporin. A virtually identical pattern is observed for formation of polyketides,218 219 whose chemistry is considered in Chapter 21. 

E represents the enzyme, S the substrate or reactant, and P the product. For a specific enzyme, only one or a few different substrate molecules can bind in the proper manner and produce a functional ES complex. The substrate must have a size, shape, and polarity compatible with the active site of the enzyme. Some enzymes catalyze the transformation of many different molecules as long as there is a common type of chemical linkage in the substrate. Others have absolute specificity and can form reactive ES complexes with only one molecular structure. In fact, some enzymes are able to differentiate between D and L isomers of substrates. 

GDP-ot-D-mannose (23) is the donor substrate for mannosyltransferases [139, 146, 338-340] and the precursor of GDP-(3-L-fucose (13) [173,197, 243, 341], Based on the work of Munch-Petersen [342, 343], only crude extracts from yeast have been used for the enzymatic synthesis of labeled and unlabeled 23 and GDP-deoxymannose derivatives (Table 4) [303-305, 307, 308, 344-346] as well as for the in situ regeneration of 23 (Table 4). Common to all these approaches is the use of chemically synthesized sugar-1-phosphates as substrates for GDP-Man PP. An obvious disadvantage of using crude yeast enzyme preparations is the poor quality of the enzyme source since only fresh cells or certain batches of baker s yeast are suitable for synthesis [304, 307], GDP-Man PP was purified from pig liver and used for the synthesis of 8-Azido-GDP-Man however, the enzyme lacks absolute specificity for GDP-Man in the pyrophos-phorylysis reaction [309]. 

These isomers and all a-thionucleotide diastereomers can be conveniently distinguished by 3iP-NMR spectrometry, as shown by Sheu et al. [23] and also by Jaffe and Cohn [38]. The Pa chemical shifts for the diastereomers of a-thionucleotides differ by 0.25-0.4 ppm, (Rp) upfield, and similar differences exist between the diastereomers of ATP/JS. 31P-NMR is probably the most positive way of assigning configuration to these nucleotides. This is because the enzymes often do not exhibit absolute specificity for one or the other isomer, and a false positive result can be obtained if too much test enzyme is used with an unknown. A typical set of spectra for a mixture of a-thionucleotides is given in Fig. 12. 

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