RAMAN SPECTRAL EVIDENCE FOR AN ANHYDRIDE INTERMEDIATE IN THE CATALYSIS OF ESTER HYDROLYSIS BY CARBOXYPEPTIDASE-A

Single crystals of carboxypeptidase A, grown at pH 7.5, have been soaked with solutions of a chromophoric substrate, the ester of p-dimethylaminobenzoic acid and L-beta-phenyllactic acid, at pH = 8. The enzyme in the crystal catalyzes the, conversion of the ester to the acid and alcohol products. During this process, pre-resonance-enhanced Raman spectra of the interior of the crystal were taken using a laser Raman microscope. In addition to the Raman bands characteristic of the substrate and the acid product, weak but reproducible bands are observed in the frequency region of 1700-1800 cm-1. Bands in the region are unique to organic acid anhydrides and indicate the accumulation of an anhydride intermediate during catalysis. A comparison of these spectra with those of model compounds designed to mimic a pure anhydride intermediate and a Zn(II)-complexed anhydride intermediate suggests the active-site Zn(II) is sometimes bound to the carbonyls of the anhydride intermediate. In an attempt to observe the intermediate in the reverse reaction, the hydrolysis products of the ester substrate were combined with the enzyme crystal, and Raman spectra were obtained from the interior of the crystal. Since the Raman spectrum taken during the forward reaction is dominated by the presence of the excess substrate while the Raman spectrum taken during the reverse reaction contains no substrate bands, the two spectra are quite different. However, there are Raman bands in the spectrum taken during the reverse direction that correlate well with the bands produced from the forward reaction and with the bands of the model compounds that are assigned to the anhydride intermediate. This indicates that the reverse reaction proceeds as far as the formation of the intermediate, but no measurable substrate is formed. Since carbonyl vibrational frequencies are very sensitive to the molecular environment, the carbonyl frequencies of the ester substrate, protonated acid product, and mixed anhydride model compounds were measured in various solvents. Plots were made of the carbonyl frequencies versus Gutmann's electron acceptor numbers of the solvents. It is shown that only the mixed anhydride can possess carbonyl frequencies in the region 1720-1800 cm-1. This appears to rule out the possibility that the bands observed from the enzyme/ligand spectra are attributable to ester substrate or acid product. These data are consistent with a mechanism that involves a mixed anhydride intermediate that becomes activated toward hydrolysis through binding of one or both carbonyls to the active-site Zn(II).