Carboxypeptidase changes

The effects of various enzymes on the activity of HPLC fractions that inhibited 3H-PCP binding were investigated. As shown in table 1, pronase (0.5 pg/ml), carboxypeptidase A (0.1 unit/ml), and trypsin (3.0 g/ml ) markedly decreased the potency of 10 n units of PCP-like activity. No significant change in activity was. seen when fractions were incubated with alpha-chymotrypsin. 

In the case of carboxypeptidase B, Shaklai et al.(2lT> compared the relative contributions to the protein phosphorescence from tyrosine and tryptophan for the apoenzyme, the zinc-containing metalloenzyme in the absence of substrate, the metalloenzyme in the presence of the substrate iV-acetyl-L-arginine, and the metalloenzyme in the presence of the specific inhibitor L-arginine. The tyrosine tryptophan emission ratio of the metalloenzyme was about a factor of four smaller than that of the apoenzyme. Binding of either the substrate or the inhibitor led to an increase in the emission ratio to a value similar to that of the apoenzyme. The change in the tyrosine tryptophan phosphorescence ratio was attributed to an interaction between a tyrosine and the catalytically essential zinc. The emission ratio was also studied as a function of pH. The titration data are difficult to interpret, however, because a Tris buffer was used and the ionization of Tris is strongly temperature dependent. In general, the use of Tris buffers for phosphorescence studies should be avoided. 

Four examples of catalytic or regulatory zinc proteins are reviewed here, and the discussion of metalloprotein function is set within the context of the metal ion and its coordination polyhedron. In the zinc enzymes carbonic anhydrase (carbonate dehydratase) II and carboxypeptidase A, the coordination polyhedron of the metal ion changes as the... 

Potts et al. 89) have shown that the 5 C-terminal residues of S-peptidfe can be removed with carboxypeptidase. The resulting derivative (residues 1-15) forms a strong complex with S-protein having full catalytic activity. It is clear from the X-ray structure that these 5 residues interact little, if at all, with any part of S-protein, and they are remote from the active site. The various changes produced in this component by synthesis and by chemical modifications are discussed later. 

Carboxypeptidase action at 25° on S-protein removes Val 124 very rapidly with no effect on the RNA activity regenerated with added S-peptide 90). Further digestion removed Ser 123 with an activity drop to 45%, but the peptide-protein binding constant changed very little. More... 

Figure 23 The conformational changes induced in carboxypeptidase on binding of the pseudosubstrate glycyltyrosine...

It may be concluded, therefore, that with samall synthetic substrates of the type Z-X-Y, where the X-Y bond is broken, the acid carboxypeptidase exhibits a preference for aromatic or carboxyl in the X position. Only limited data are available on the effect of changing the X and Y groups on the side chain specificity of the enzyme, but it would appear that this conclusion holds for substrates in which the X group is changed from tyrosine or phenylalanine to glycine. 

The mechanism of action of carboxypeptidase has been subject to analysis in terms of ALPH by Deslongchamps (1983, p. 351). Unfortunately, the balance of evidence is now that the covalency changes so analysed probably do not take place, and that zinc proteinases work by a substantially different mechanism to that assumed by Deslongchamps in his stereoelectronic analysis. 

Generally speaking, the role of the enzyme consists of the selective and specific attraction of substrate and the highly efficient catalysis. Every enzyme has its own characteristic feature for example, the specificity in the binding and a charge-relay action in the catalysis in a-chymotrypsin, the contribution of the imidazole moiety as an electron donor to the electrophilicity of zinc ion in carboxypeptidase, the change in the spin state and the reactivity of the transition metal ion by the coordination of the imidazole in the hemochrome. These typical characteristic features are the result of the cooperative actions of the constituents. 

Electrophilic action in enzymes is carried out by metal ion or coenzyme. The metal ion which is a component of the active center is bound to imidazole moiety in histidine by chelation. The typical enzymes are carboxypeptidase and decarboxylase, etc. A change in electronic structure of the metal ion by the chelation plays an important role in the catalysis. The catalytic activity of carboxypeptidase can be illustrated as follows (11),... 

Although it is true that abnormal proteins increase with age, most of them are a result of posttranslational changes. An example is the various isoforms of creatine kinase (CK). Here, the major isoenzyme, CK-MM (isoform CK-33), is normally synthesized in the heart and skeletal muscle. However, after its release into the circulation, carboxypeptidase hydrolyzes the terminal lysine from one of the M-peptides to form CK-32. Subsequent hydrolysis of the terminal lysine from the second M-peptide produces the third isoform, CK-3i (W8). Numerous similar posttranslational proteins are produced. Hence, the presence of abnormal proteins per se does not support this aging theory. 

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