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. 

CoA dehydrogenase shows absolute specificity for the L-hydroxyacyl isomer of the substrate (Figure 24.16). (o-Hydroxyacyl isomers, which arise mainly from oxidation of unsaturated fatty acids, are handled differently.)... 

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. 

One of the terms for describing enantiomer composition is optical purity. It refers to the ratio of observed specific rotation to the maximum or absolute specific rotation of a pure enantiomer sample. For any compound for which the optical rotation of its pure enantiomer is known, the ee value may be determined directly from the observed optical rotation. 

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... 

The use of bromcresol green for the measurement of albumin has been criticized on several counts. There is a tendency for the protein-dye complexes to precipitate at pH 4.2, which is very near the iso-ionic pH of albumin. It is claimed that the method is not absolutely specific for albumin and particularly with serum samples shows a positive bias in results. There is also some variability in the intensity of the colour produced with albumins from different sources, a fact which makes the choice of the standard material important. 

The use of the term marker is often misleading because it can be interpreted to imply an absolute specificity that does not reflect the dynamic nature of intracellular compartments. It is important to remember that the predominant location of these markers reflects a balance of trafficking pathways into and out of each compartment. Marker can be internalized not only by one but by different mechanisms. Even SV40, which is known to be a specific marker for caveolae uptake, is now also described as entering the cells via a different mechanism (121). Therefore, not just one but several inhibitors or markers for each mechanism should be applied and all results should be taken into account before reaching a conclusion. 

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. 

A method by Berger[12] utilized the stereoselective hydrolysis of diastereomeric peptides by leucine aminopeptidase. Z-L-Ala-D-Ala-OH was coupled to an all L-Ala peptide such as l-Ala-L-Ala-ONbz. Epimerization during coupling resulted in the formation of a small amount of L-Ala-L-Ala-L-Ala-L-Ala after deprotection, and since the peptidase has an absolute specificity for the all-L peptide, only the epimerized product was hydrolyzed. Quantification of the degradation products gave the extent of epimerization. These classical procedures, however, are specific to the particular coupling reaction under consideration and the results may not be fully applicable to all systems. Furthermore, they give no direct information about the rate of racemization. 

The maximum specific rotation [a]mal (absolute specific rotation) of one of the pure enantiomers must be known with certainty and it must be confirmed by an independent method. 

The 3-receptor-blocking drugs differ in their relative affinities for and 32 receptors (Table 10-1). Some have a higher affinity for 3 than for 32 receptors, and this selectivity may have important clinical implications. Since none of the clinically available 3-receptor antagonists are absolutely specific for receptors, the selectivity is dose-related it tends to diminish at... 

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. 

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