Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism.

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.