Carboxypeptidase-Catalyzed Hydrolysis

An amino acid sequence is ambiguous unless we know the direction m which to read It—left to right or right to left We need to know which end is the N terminus and which IS the C terminus As we saw m the preceding section carboxypeptidase catalyzed hydrolysis cleaves the C terminal ammo acid and so can be used to identify it What about the N terminus ... 

Carboxypeptidase catalyzed hydrolysis can be used to identify the C terminal ammo acid The N terminus is determined by chemical means One reagent used for this purpose is Sanger s reagent 1 fluoro 2 4 dimtrobenzene (see Figure 27 9)... 

An important difference between thermolysin and carboxypeptidase leads to the major uncertainty in the mechanism of carboxypeptidase. This difference is that the catalytic carboxylate of carboxypeptidase is far more sterically accessible. The crucial question is whether or not the carboxypeptidase-catalyzed hydrolysis of peptides proceeds via general-base catalysis, as in equation 16.26, or via nucleophilic catalysis, as in 16.27. Early kinetic work concentrated on establishing the participation of the various groups in catalysis. 

A-4. The carboxypeptidase-catalyzed hydrolysis of a pentapeptide yielded phenylalanine (Phe). One cycle of an Edman degradation gave a derivative of leucine (Leu). Partial hydrolysis yielded the fragments Leu-Val-Gly and Gly-Ala among others. Deduce the structure of the peptide. 

M. Perryman, D. Knell, and R. Roberts. Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine mechanism for in vivo production of multiple forms of creatine kinase in plasma. Clin. Chem. 30 662-664 (1984). 

Fig. 12. Enzyme tryptophan (A) and substrate dansyl (B) fluorescence during the time course of zinc carboxypeptidase catalyzed hydrolysis of DNs-Gly-L-Phe. Equal volume solutions of substrate and of enzyme, both 2.5 x 10 M, in 1 M NaCl-0.02 M Tris, pH 7.5, 25°, were mixed and the fluorescence of either tryptophan (A) or dansyl (B) was measured as a function of time under stopped-flow conditions in parallel samples, as shown by the oscilloscope tracings. Excitation was at 285 nm. Scale sensitivities for (A) and (B) are 100 mV/div. The existence of the E S complex is signalled either by (A) the suppression of enzyme tryrptophan fluorescence (quenching by the dansyl group) or (B) enhancement of the substrate dansyl group fluorescence (energy transfer from enzyme trjqrtophan). Taken from Ref. (182) with permission...

Amide Bond Formation Between a Carboxylic Acid and an Amine Using N,N -Dicyclohexylcarbodiimide 1152 Carboxypeptidase-Catalyzed Hydrolysis 1162... 

THE MECHANISM The mechanism shown outlines the major stages in carboxypeptidase-catalyzed hydrolysis of a peptide in which the C-terminal amino acid is phenylalanine. Proton transfers accompany stages 2 and 3 but are not shown. Only the major interactions of the substrate with the carboxypeptidase side chains are shown although others may also be involved. 

Stage 2 Water adds to the carbonyl group of the peptide bond. The rate of this nucleophilic addition is accelerated by coordination of the carbonyl oxygen to Zn and/or to one of the N—protons of Arg-127 (not shown). The product is a tetrahedral intermediate stabilized by coordination to zinc. Stabilization of the tetrahedral intermediate may be the major factor for the rapid rate of the carboxypeptidase-catalyzed hydrolysis. 

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