Carboxypeptidase enzymes related

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

Zn+2, Mn+2, Fe+2, Cd+2, Co+2, and Ni+2, although diminished activity results (35, 53). Fundamentally the enolase and aconitase reactions are closely related, since the net result of both is the addition or subtraction of a molecule of water. In spite of this similarity, the metal ions associated with these two reactions are very different. It is one of the puzzling aspects of metalloenzyme chemistry that every enzyme has a different metal ion specificity. Each of these enzymes is associated in its natural state with a specific metal ion, which differs from enzyme to enzyme. It is possible to remove the naturally occurring metal from many of these enzymes and to reactivate them by the addition of other metals, as has been shown in the case of carboxypeptidase. The order in which the various metal ions fall in their ability to activate the different enzymes again varies from enzyme to enzyme. 

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

The acid carboxypeptidase from A. saitoi releases the carboxyterminal phenenylalanine-amide (-Phe-NH2) from the carboxy-terminal amidated peptides, such as gastrin-related peptide (/-amyloxycarbonyl (Aoc)-Trp-Met-Asp-Phe-NH2, Aoc-WMDF-NH2) and molluscan cardioexcitatoiy neuropeptide (Phe-Met-Arg-Phe-NH2, FMRF-NH2) [86], The summarized data are shown in Table 12. When gastrin-related peptide was used as a substrate, the enzyme acted only as a carboxyamidase, because of the presence of the hydrophobic amino acid residue, tryptophan, in the P3 [12] position. 

Cysteine proteases are so called because of a critical cysteine involved (together with an adjacent histidine) in the catalytic mechanism. Cysteine proteases include papain-related proteases, calpain-related proteases and the caspases. Papain-like cysteine proteases include the plant enzymes actinidin, aleurain, bromelain, caricain, chymopapain, ficin and papain and the lysosomal cathepsins B, C, H, K, L and S. Cathepsin C is multimeric (MW -200,000), but the other papain-related proteases are monomeric with MWs of about 20,000-35,000. While cathepsin C is a dipeptidyl aminopeptidase, the other enzymes are endopeptidases. Cathepsin B is an endopeptidase and a dipeptidyl carboxypeptidase. Cathepsin H is an endopeptidase and an aminopeptidase. In higher animals, cathepsin B generates peptides from antigens for presentation to T cells by the major histocompatibility... 

There are no significant differences between ethyl and methyl esters concerning synthesis or cleavage. Related protocols of the methyl esters (see Section 2.2.1.1.1.1) are, therefore, applied to the ethyl esters. The usefulness of ethyl esters is somewhat limited by the difficulties encountered in their saponification. Hydrolysis with alkali is feasible, but ethyl esters are less sensitive to nucleophilic attack than methyl esters. Aminolysis and hydrazinolysis as well as cleavage of the alkyl-oxygen bond with lithium iodide in pyridineb l proceed several times slower in the case of ethyl esters. Mild enzyme-catalyzed hydrolysis by trypsin and chymotrypsin,t 2° 2 1 or by carboxypeptidase remains an attractive alternative. 

Zinc is used by a great number of enzymes and proteins, whereas copper seems lobe limited to only a few functions. The most thoroughly studied zinc metailoen-zymes of mammals are carbonic anhydrase, carboxypeptidase A and related pep-... 

Two important zinc metalloenzymes in protein digestion are the pancreatic carboxypeptidase A and B. A loss of activity of the pancreatic carboxyeptidase A in zinc deficiency is a consistent finding (91), According to some investigators, within two days of institution of zinc-deficient diet in rats, the enzyme lost 24% of its activity, and within three days of dietary zinc repletion, the activity of pancreatic carboxypeptidases is restored to normal levels (91). These results indicate that the level of food intake has no influence and that a decreased activity of carboxypeptidase A in the pancreas was related specifically to a dietary lack of zinc. As in the case of the alkaline phosphatase, the amount of carboxypeptidase A apoenzyme present appears to be diminished in zinc-deficient pancreas. 

Zinc content and the activity of the enzyme are directly related. The metal is aggregated in the fractions of highest activity (Table VII). Throughout recrystallization, both the enzymatic activity and the zinc content remain at a constant level. The presence of all other metals is apparently unrelated to activity through fractionation. Thus, constant zinc/ protein, zinc/activity, activity/protein ratios are achieved with purification of carboxypeptidase. It is unlikely that the metal is a chance contaminant, since the molar zinc/protein ratio of the crystalline enzyme is an integral number, i.e., 1. 

Based on the crystallographic data, detailed mechanisms for the carboxypeptidase A enzymic reaction have been proposed. These mechanisms and recent work relating to them have been reviewed.Although probably correct in general, these mechanistic conclusions are based on the assumption that the kinetic and chemical properties are conserved on crystallization. In general coordination chemistry examples abound where the structures of species in the crystd and in solution are markedly different and indeed it has been shown that the detailed kinetics of carboxypeptidase A solutions differ from those of the enzyme crystals. It has been suggested that different conformations of the active site exist in the two physical states,Detailed kinetic studies on crystals over a range of enzyme concentrations, substrate concentrations and crystal sizes have been carried out and the results interpreted in terms of a recent theory for insolubilized enzymes. The marked differences... 

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