Carboxypeptidase intermediates

The reactivity of the coordinated, deprotonated nucleophile is typically intermediate between that of the un-ionized and ionized forms of the nucleophile. Carboxypeptidase (Chapter 5) contains an active site Zn, which facilitates deprotonation of a water molecule in this manner. 

Fig. 31. Mechanistic proposal for peptide hydrolysis catalyzed by carboxypeptidase A (Christianson and Lipscomb, 1989). (a) The precatalytic Michaelis complex with substrate carbonyl hydrogen bonded to Arg-127 allows for nucleophilic attack by a water molecule promoted by zinc and assisted by Glu-270 (an outer-sphere C==O Zn interaction is not precluded), (b) Tbe stabilized tetrahedral intermediate collapses, with required proton donation by Glu-270 (Monzingo and Matthews, 1984) Glu-270 may play a bifunctional catalytic role (Schepartz and Breslow, 1987), which results in the product complex (c). [Reprinted with permission from Christianson, D. W., Lipscomb, W. N. (1989) Acc. Chem. Res. 22,62-69. Copyright 1989 American Chemical Society.]...

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

Human proinsulin has been synthesized in homogeneous solution from 11 protected fragments using azide coupling.1151 The difficulties with insoluble intermediates were sufficiently overcome to allow the 86-residue peptide to be synthesized. The product was de-protected, converted into the 5-sulfonate, and then reduced and reoxidized to form the three disulfide bonds. The product was extensively purified and analyzed, and shown to be pure proinsulin. This product could then be converted into insulin by the use of endopeptidases I and II from pancreatic (3-cell granules, together with carboxypeptidase H, which removed the four basic residues 31, 32, 64, and 65, and split out C-peptide.1 6 ... 

There is a third region of a protein that is neither on the surface nor in the interior but that is in a cleft. Such regions are often associated with enzyme action and examples show they have (1) intermediate mobility (e.g., tryptophan 62 of lysozyme or the tyrosine of carboxypeptidase) and (2) unfavorable energetics of exposed groups—the entatic state. 

Thiosulfate cyanide sulfurtransferase symmetry in 78 TTiiouridine 234 Three-dimensional structures of aconitase 689 adenylate kinase 655 aldehyde oxido-reductase 891 D-amino acid oxidase 791 a-amylase, pancreatic 607 aspartate aminotransferase 57,135 catalytic intermediates 752 aspartate carbamyltransferase 348 aspartate chemoreceptor 562 bacteriophage P22 66 cadherin 408 calmodulin 317 carbonic acid anhydrase I 679 carboxypeptidase A 64 catalase 853 cholera toxin 333, 546 chymotrypsin 611 citrate synthase 702, 703 cutinase 134 cyclosporin 488 cytochrome c 847 cytochrome c peroxidase 849 dihydrofolate reductase 807 DNA 214, 223,228,229, 241 DNA complex... 

Little is known about the regulation mechanisms of the synthesis of complex carbohydrate in plants, through lipid intermediates. However, partial evidence indicates that lipid-mediated glycosylation in proteins could be a regulatory step. When glycosylation of carboxypeptidase Y is inhibited... 

One possible mechanism for the hydrolysis of peptides or esters by carboxypeptidase A involves two steps with an anhydride (acyl-enzyme) intermediate.418 In the first step, the zinc(II) activates the substrate carbonyl group towards nucleophilic attack by a glutamate residue, resulting in the production of a mixed anhydride (127). Breakdown of the anhydride intermediate is rate determining with some substrates.419 An understanding of the chemistry of metal ion effects in anhydride hydrolysis is therefore of fundamental importance in regard to the mechanism of action of the enzyme. Until recently there have been few studies of metal ion-catalysed anhydride solvolysis. 

The mechanism of action of carboxypeptidase has also been much studied recently by the techniques of cryoenzymology, which have allowed the identification and characterization of reaction intermediates.519,520 This has shown the presence of two intermediates during the hydrolysis of both peptides and esters. In conjunction with chemical evidence, this work demonstrates that there is no acyl intermediate in either peptide or ester hydrolysis, and that these two substrates form different metallo intermediates and are hydrolyzed through different mechanisms. 

It can therefore be concluded that the sites of secondary substrate recognition of the enzyme which impose a strain on the substrate and cause a geometrical distortion of the tetrahedral intermediate, are an obligatory part of the catalytic action of carboxypeptidase A which takes place under stere-oelectronically controlled conditions. 

Specific ester substrates are also hydrolyzed with carboxypeptidase A. For instance, Makinen, Fukuyama, and Kuo (27) have recently studied the enzymic hydrolysis of 0-(trans-p-ch1orocinnamoyl)-L-B-phenyl actate (CICPI.) (47),and the spin labeled nitroxide ester substrate 0-3-(2,2,5,5-tetramethylpyrrol-inyl-l-oxyl)-propen-2-oyl-L-B-phenyllactate (TEPOPL) (48). They have shown that these reactions take place via the formation of a covalent intermediate (the mixed anhydride) which can be isolated under subzero temperature conditions. The hydrolysis of CICPL and TEPOPL catalyzed by carboxypeptidase A is consequently governed by the rate-limiting breaking of the acyl-enzyme. 

Stable compounds which resemble the transition-state structure of a substrate in an enzymatic reaction are expected to behave as potent reversible inhibitors (1 ). Based on the X-ray crystallographic structure of the active site of carboxypeptidase A (CPA) (2), a mechanism was proposed in which a water molecule adds directly to the scissile carbonyl group of the substrate to give the tetrahedral intermediate 1, which collapses to products (3). We proposed to mimic this tetrahedral intermediate, similar to the transition state, with the stable tetrahedral phosphonic acid derivatives 2,... 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

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