The diffusion-dependent kinetic properties of the yeast glyoxalase I reaction have been measured by means of viscosometric methods. For the glyoxalase-I-catalyzed isomerization of glutathione (GSH)-methylglyoxal thiohemiacetal to S-D-Iactoylghtathione, the k,,,lK, (3.5 X 10" M-' s-', pH 7, 25 "C) undergoes a progressive decrease in magnitude with increasing solution viscosity, using sucrose as a viscogenic agent. The viscosity effect is unlikely to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, as the magnitude of k,.,,lK,, for the slow substrate GSH-t-butylglyoxal thiohemiacetal (3.5X1O3M-' s-', pH 7, 25OC) is independent of solution viscosity. Quantitative treatment of the data by means of the Stokes-Einstein diffusion law suggests that catalysis will be about 50% diffusion limited under conditions where [substrate] < K,; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. In a related study, the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing human erythrocytes are estimated to be in the nanomolar concentration range, on the basis of published values for the activities of glyoxalase I and glyoxalase I1 in lysed erythrocytes and the steady-state rate of formation of D-lactate in intact erythrocytes. This is consistent with a model of the glyoxalase pathway in which the enzyme-catalyzed steps are significantly diffusion limited under physiological conditions.

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