Hydrogen bonding carboxypeptidase

U. H. Mortensen, S. J. Remington, K. Breddam, Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y. A Hydrogen Bond Network Stabilizes the Transition State by Interaction with the C-Terminal Carboxylate Group of the Substrate , Biochemistry 1994, 33, 508-513. 

Chemical reactivity and hydrogen bonding 320 Proton-transfer behaviour 321 Intramolecular hydrogen-bond catalysis 344 Enzyme catalysis and hydrogen bonding 354 Chymotrypsin 354 Thermolysin 355 Carboxypeptidase 355 Tyrosyl tRNA synthetase 356 Summary 366 Acknowledgements 367 References 367... 

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.]...

Carboxypeptidase A"" (CPA, EC 3.4.17.1) is a proteolytic enzyme that cleaves C-terminal amino acid residues with hydrophobic side chains selectively. Several X-ray structures are available" The active site of CPA consists of a hydrophobic pocket (primary substrate recognition site) that is primarily responsible for the substrate specificity, a guanidinium moiety of Argl45 that forms hydrogen bonds to the carboxylate of the substrate, and Glu270, whose carboxylate plays a critical role, functioning either as a nucleophile to attack the scissUe carboxamide carbonyl carbon of the substrate or as a base to activate the zinc-bound water molecule, which in turn attacks the scissile peptide bond ". However, semiempirical calculations had shown that the direct attack 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. 

Figure 25-19 Steps in a possible mechanism of carboxypeptidase action, (a) The substrate is shown complexed to the enzyme surface through X, Y, Z, and W X is a nonpolar pocket Y is a hydrogen bond, possibly from OH of Tyr 248 Z is the prosthetic group, Zn and W is an...

Because internal water molecules are in mostly apolar environments, their hydrogen bonds are often strong and well defined. The average O- -O distance is 2.89 0.21 A for the internal water molecules in lysozyme, carboxypeptidase, cytochrome c, actinidin, and penicillopepsin. As with the small molecule crystal structures, the water molecules are involved in three or four hydrogen bonds, with 48% engaged in three and 37% in four interactions. 

Hydrogen-bond partners for internal water molecules. In the five proteins lysozyme, carboxypeptidase, cytochrome c, actinidin and penicillopepsin (Table 19.1), the protein groups (numbers in parentheses) which are bonded to internal water molecules are the main-chain C=0 (75), N-H (38), and side-chain atoms... 

Bovine carboxypeptidase A was only the third protein, after myoglobin and lysozyme, to have its three-dimensional structure solved at high resolution. The active site zinc is bound to His-69, Glu-72, and His-196 (Figure 12.5), and to a water molecule. The zinc-bound water molecule is itself hydrogen bonded to Glu-270. Despite extensive experimental data, the mechanism of carboxypeptidase still remains controversial. Two major... 

N and C termini of the peptide are connected via a linker to form a cyclic peptide. The linker can be cleaved by esterase to release the peptide. The cyclic peptide pro-drug formation increases the intramolecular hydrogen bonding and lowers the hydrogen-bonding potential to water molecules as solvent. In addition, the lipophi-licity of the cyclic prodrug increases, which shifts its transport from paracellular to transcellular.48 It has also been reported that cyclic peptides are less susceptible to amino- and carboxypeptidases than linear peptides because the amino and carboxy terminals are protected from these enzymes.44... 

Figure 6 Amino acids in the active site of carboxypeptidase N. The dipeptide glycyl-L-tyrosine is docked into the active site. The active site zinc is coordinated to His 69, Glu 72, and His 196. Arg 145, Asn 144, and "tyr 248 provide specificity for substrates bearing a free terminal carboxylate. Gin 255 forms a hydrogen bond with the side chain of the carboxy-terminal arginine or lysine. Glu 270 promotes the nucleophilic attack of a water molecule at the scissile peptide bond.

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