ELUCIDATION OF THE STEREOCHEMISTRY OF THE CARBOXYPEPTIDASE-A CATALYZED ENOLIZATION OF 2-BENZYL-3-PARA-METHOXYBENZOYLPROPIONATE, A KETONE SUBSTRATE

Carboxypeptidase A³catalyzes hydrogen-deuterium exchange with retention of configuration at the activated methylene group of (-)-2-benzyl-3-p-methoxybenzoylpropionic acid, (-)-l, a ketonic analogue of the enzyme's ester and peptide substrates. However, (+)-l does not undergo the corresponding exchange process even though it is bound to the enzyme slightly more tightly than the - isomer.1H NMR measurements at 270 MHz show that the signals for the diastereotopic protons on the activated methylene group of 1 appear separately at 2.98 (Ha) and 3.33 ppm (Hb) and that exchange occurs only at the Ha position of (-)-l. Degradation of the enantiomers of 1 by Baeyer-Villiger oxidation and subsequent hydrolysis to give the corresponding isomers of 2-benzylsuccinic acid showed that (-)-l has the R configuration at the chiral methine group. cis- and trans-2-benzyl-4-p-methoxyphenyl-γ-butyrolactones containing hydrogen and/or deuterium at the 3-methylene position were prepared by sodium borohydride reduction of 1 and various of its deuterated forms, followed by cyclization using dicyclohexylcarbodiimide. Comparison of the1H NMR spectra of the lactones with those of the cis-2-benzyl-4-p-methoxyphenyl-7-butyrolactones produced by the cyclization of 1 and 1-d2(substituted with deuterium at the 3 position) with acetic anhydride, followed by reduction with hydrogen over platinum oxide, showed that the Ha proton on the activated methylene group is in the pro-R configuration. The stereochemical results obtained are consistent with the hypothesis that (-)-l binds in a manner similar to that which has already been postulated for reactive peptide and ester substrates and that the γ-carboxylate moiety of Glu-270 is the functional group in the enzyme which abstracts the Hahydrogen from the 3 position of the ketone substrate. © 1979, American Chemical Society. All rights reserved.