STEREOSPECIFICITY OF SUBSTRATE USAGE BY GLYOXALASE-I - NUCLEAR-MAGNETIC-RESONANCE STUDIES OF KINETICS AND HEMITHIOACETAL SUBSTRATE CONFORMATION

The specificity of glyoxalase 1 for the diastereomers of its hemithioa cetal substrate [which forms spontaneously between an alpha-keto aldeh yde and reduced glutathione (GSH)] was investigated by exploiting the differences between their H-1 NMR spectra at pH 4.4. The H-1 NMR spect ra of the hemithioacetals of glutathione with phenylglyoxal or methylg lyoxal were assigned with the aid of conventional decoupling and two-d imensional NMR spectroscopic techniques. The rate of interconversion o f the diastereomers was determined at 30-degrees-C from the results of an inversion-transfer technique and found to be 0.30 +/- 0.04 s-1 (+/ - sd) in the case of phenylglyoxal and 0.15 +/- 0.02 s-1 in the case o f methylglyoxal. Stereopreference of the enzyme was tested by the addi tion of large amounts of yeast glyoxalase 1 to a reaction mixture; gly oxalase 1 preferentially operated on one diastereomer of the phenylgly oxal hemithioacetal but the diastereomers of methylglyoxal appeared to be operated upon indiscriminately. From computer models of the kineti cs of possible reaction schemes, a mechanism involving glyoxalase 1 ca talysis of both diastereomers of the hemithioacetals was shown to be t he most consistent with the experimental data. Estimates of internucle ar distances in the diastereomers, obtained from 2D NMR spectra were u sed in ''dynamical simulated annealing'' calculations to generate like ly structures of the substrates. Relative ring-current shifts obtained from ID NMR spectra were used, together with a ring-current shift alg orithm, to select structures with compatible conformations. We conclud e that the rate of conversion of substrate by the enzyme is dependent upon the overall conformation of the substrate molecule, rather than m erely its stereochemical configuration (R or S).