The pH-Rate Profile for the Hydrolysis of a Peptide Bond

Abstract

The rate of hydrolysis of N -(phenylacetyl)glycyl- d -valine (PAGV), an acyclic penicillin G analogue, at pH 0, 1, 3, 5, 7, 9, 11, 13, and 14 has been measured at 37 °C and a pH-rate profile constructed. At each pH, hydrolysis of both the (phenylacetyl)glycyl amide bond and glycyl- d -valine peptide bond was monitored. At pH 3, 5, 7, 9, and 11, the hydrolysis products glycyl- d -valine and d -valine were derivatized with naphthalene-2,3-dialdehyde in the presence of cyanide; the resultant 1-cyano-2-substituted-benz[ f ]isoindole (CBI) derivatives, which are highly fluorescent, were then quantified using reverse-phase HPLC. The hydrolysis reactions were explicitly shown to be first-order in peptide concentration at pH 5 and 9, and all rates were shown to be independent of the buffer concentration. The rates at pH 0, 1, 13, and 14 were measured in 1 M DCl, 0.1 M DCl, 0.1 M NaOD, and 1 M NaOD, respectively, and the hydrolysis products were detected by 1 H NMR. The first-order rate constants obtained from the above reactions were fit to the general equation k = k H 2 0 + k H 3 0 + [H 3 O + ] + k OH − [OH - ] to yield the following results: for hydrolysis of the (phenylacetyl)glycyl bond, k H 2 O = (9.05 ± 6.36) × 10 - 11 s - 1, k H 3 O + = (1.60 ± 1.04) × 10 - 6 M - 1 s - 1, and k OH − = (1.11 ± 0.73) × 10 - 6 M - 1 s - 1; and for hydrolysis of the glycyl- d -valine bond, k H 2 O = (8.23 ± 4.33) × 10 - 11 s - 1, k H 3 O + = (1.67 ± 0.80) × 10 - 6 M - 1 s - 1, and k OH − = (1.16 ± 0.56) × 10 - 6 M - 1 s - 1 . At pH 7, the hydrolysis of both the (phenylacetyl)glycyl amide bond and glycyl- d -valine peptide bond is dominated by k H 2 O . The corresponding half-life for (phenylacetyl)glycyl bond hydrolysis is 243 years (with a range of 143−817 years within experimental error), while that for glycyl- d -valine bond hydrolysis is 267 years (with a range of 175−564 years).