Diffusion‐Dependent Kinetic Properties of Glyoxalase I and Estimates of the Steady‐State Concentrations of Glyoxalase‐Pathway Intermediates in Glycolyzing Erythrocytes

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 ‐lactoylglutathione, the k cat / K m (3.5 × 10 6 M −1 S −1 , pH7, 25°C) undergoes a progressive decrease in magnitude with increasing solution viscosity, using sucrose as a visco‐genic 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 cat / K m for the slow substrate GSH‐ t ‐butylglyoxal thiohemiacetal (3.5 × 10 3 M −1 s −1 , pH 7, 25°C) 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 m ; 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 II 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.