Zinc, in carboxypeptidase

P-CD derivatives have also been used to catalyze the hydrolysis of esters, mimicking the mechanism employed by carboxypeptidases. Carboxypepti-dase A is a metalloenzyme that is much more effective than chymotrypsin in hydrolyzing amide bonds. Zinc in carboxypeptidase A has a typical... 

The presence of zinc in carboxypeptidase would appear to elucidate the problem. 

In metalloproteins, the metal cofactor may be removed and replaced by a heavier atom with similar chemistry. The zinc in insulin was successfully replaced by cadmium or lead, the zinc in carboxypeptidase and carbonic anhydrase by mercury, the zinc in thermolysin by lanthanide ions or strontium or barium and the calcium in staphylococcal nuclease by barium. Success has most usually been achieved by soaking the crystals in a chelating agent and subsequently diffusing in the heavier atom. 

Fig. 1. Coordination of zinc in carboxypeptidase enzyme-substrate complex (See Ref. 15).

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

Another contrast between the zinc proteases and the carbonic an-hydrases concerns the zinc coordination polyhedron. The carbonic an-hydrases ligate zinc via three histidine residues, whereas the zinc proteases ligate the metal ion through two histidine residues and a glutamate (bidentate in carboxypeptidase A, unidentate in thermolysin). Hence, the fourth ligand on each catalytic zinc ion, a solvent molecule, experiences enhanced electrostatic polarization in carbonic anhydrase II relative to carboxypeptidase A. Indeed, the zinc-bound solvent of carbonic anhydrase II is actually the hydroxide anion [via a proton transfer step mediated by His-64 (for a review see Silverman and Lindskog, 1988)]. 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

The four types of nickel-containing enzymes are quite distinct in the coordination sites and catalytic function of the nickel centers. In urease, the nickel appears to be bound to oxygen and nitrogen ligands and appears to remain as Ni(II), a state which favors octahedral or square-planar coordination. The function of nickel in this unique case may be analogous to that of zinc in other hydrolases such as carboxypeptidase. 

In 1940 carbonic anhydrase was isolated from mammalian erythrocytes the protein was shown to contain 0.33% zinc. In 1955 carboxypeptidase became the second zinc enzyme to be reported. About 20 zinc metalloenzymes have since been studied in great detail, and about 60—70 await complete characterization. 

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. 

Fig. 37. Diagram representing the environment of the catalytic zinc in horse liver alcohol dehydrogenase, human carbonic anhydrase, thermolysin and carboxypeptidase A. Positions occupied by the substrate (S) and proton-abstracting group (PA) are indicated. The angles subtended by the liganding protein atoms at the zinc atom are shown to the right, and their sum (2) is shown extreme right. From the work of Rossmann and colleagues [ 164].

In carboxypeptidase, which cleaves peptide linkages (and also esters), there is first a complex in which the peptide carbonyl oxygen atom coordinates to the zinc ion and then things proceed (at least approximately) as shown in Fig. 15-8. These two mechanisms involve, in somewhat different ways, the electrophilidty of the Zn2+ ion, and it is known or believed that it is this property of the Zn2+ ion, exercised in one way or another, that is critical in all other enzymes having Zn2+ at the active site. 

Figure 9 Schematic of inhibitory zinc-binding site in carboxypeptidase A...

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