In carboxypeptidase

An artificial metalloenzyme (26) was designed by Breslow et al. 24). It was the first example of a complete artificial enzyme, having a substrate binding cyclodextrin cavity and a Ni2+ ion-chelated nucleophilic group for catalysis. Metalloenzyme (26) behaves a real catalyst, exhibiting turnover, and enhances the rate of hydrolysis of p-nitrophenyl acetate more than 103 fold. The catalytic group of 26 is a -Ni2+ complex which itself is active toward the substrate 1, but not toward such a substrate having no metal ion affinity at a low catalyst concentration. It is appearent that the metal ion in 26 activates the oximate anion by chelation, but not the substrate directly as believed in carboxypeptidase. 

Carbon atom, 4. See also Atomic orbitals Carbon dioxide hydration, 197-199. See also Carbonic anhydrase Carbonic anhydrase, 197-199,200 Carbonium ion transition state, 154, 159 Carboxypeptidase A, 204-205 Catalysis, general acid, 153,164,169 in carboxypeptidase A, 204-205 free energy surfaces for, 160, 161 in lysozyme, 154... 

Fig. 10. Examples of coordination geometry in the entatic state (34) where the Lewis acidity of Zn(II) in its metalloprotein is considerably altered as compared with that for (a) the Zn2t aquo ion (30), (b) Zn(II) in alcohol dehydrogenase, (c) Zn(II) in carbonic anhydrase, and (d) Zn(II) in carboxypeptidase. Redrawn after Ref. (22).

Scheme 1. Cooperativity in carboxypeptidase catalysis of amide hydrolysis...

FIGURE 18-12 The/af//af mutation in carboxypeptidase E (CPE) leads to secretion of proinsulin, not mature insulin, and results in diabetes. The S202P mutation within CPE results in degradation of the enzyme and defective insulin processing in the fat/fat heterozygous mouse. LDCV, large dense-core vesicle. 

N. Zisapel, N. Shaklai, and M. Sokolovsky, Metal-tyrosyl interaction in carboxypeptidase Phosphorescence studies, FEBS Lett. 51, 262-265 (1975). 

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)]. 

In carboxypeptidase differences of opinion between crystallographic and solution studies seem to be becoming resolved into a matter of... 

In both carboxypeptidase A and thermolysin the active site Zn2+ is chelated by two imidazole groups and a glutamate side chain (Fig. 12-16). In carboxypeptidase A, Arg 145, Tyr 248, and perhaps Arg 127 form hydrogen bonds to the substrate. A water molecule is also bound to the Zn2+ ion. The presence of the positively charged side chain of Arg 145 and of a hydro-phobic pocket accounts for the preference of the enzyme for C-terminal amino acids with bulky, nonpolar side chains. The Zn2+ in thermolysin is also bound to two imidazole groups and that in D-alanyl-D-alanyl carboxypeptidase to three. 

One obvious role for metals in metalloenzymes is to function as electrophilic catalysts, stabilizing the negative charges that are formed. In carboxypeptidase... 

The catalytic glutamate residue (Glu-143) is located at the bottom of a narrow cleft, where it is bound to a water molecule and cannot approach the substrate as readily as can Glu-270 in carboxypeptidase. 

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

Modifications involving Glu-270 and Arg-145 have not given such positive results. However, modification of arginyl residues in carboxypeptidase by diacetyl results in loss of peptidase activity. 

Metal binding in procarboxypeptidase A is weaker than in the active enzyme ( 107), Table 7). It was proposed that the bonding involves sulfur and a weaker ligand than N (107). In view of the present concept of the chelating site in carboxypeptidase, further studies of the zymogen are necessary. In that connection, the cobalt complex should be valuable. 

In carboxypeptidase A [52, 53], the active-site Zn(n) ion plays essential catalytic roles and the guanidinium of Arg-145 recognizes the carboxylate anion of the substrates, thus making the enzyme an exopeptidase. Important features of carboxypeptidase A reproduced by 11 include the essential catalytic action of a metal ion and participation of a guanidinium group in substrate recognition, so that this polymer biocatalyst hydrolyzes unactivated amides, and exhibits selectivity toward amide bonds adjacent to carboxylate groups in the substrate. 

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