DIFFUSION-DEPENDENT KINETIC-PROPERTIES OF GLYOXALASE-I AND ESTIMATES OF THE STEADY-STATE CONCENTRATIONS OF GLYOXALASE-PATHWAY INTERMEDIATESIN GLYCOLYZING ERYTHROCYTES

The diffusion-dependent kinetic properties of the yeast glyoxalase I r eaction have been measured by means of viscosometric methods. For the glyoxalase-I-catalyzed isomerization of glutathione (GSH)-methylglyoxa l thiohemiacetal to S-D-lactoylglutathione, the k(cat)/K-m (3.5X10(6) M(-1) s(-1), pH 7, 25 degrees C) undergoes a progressive decrease in m agnitude with increasing solution viscosity, using sucrose as a viscog enic agent. The viscosity effect is unlikely to be due to a sucrose-in duced change in the intrinsic kinetic properties of the enzyme, as the magnitude of k(cat)/K-m for the slow substrate GSH-t-butylglyoxal thi ohemiacetal (3.5X10(3) M(-1) s(-1), pH 7, 25 degrees C) is independent of solution viscosity. Quantitative treatment of the data by means of the Stokes-Einstein diffusion law suggests that catalysis will be abo ut 50% diffusion limited under conditions where [substrate] much less than K-m; the encounter complex between enzyme and substrate partition s nearly equally between product formation and dissociation to form fr ee enzyme and substrate. In a related study, the steady-state concentr ations of glyoxalase-pathway intermediates in glycolyzing human erythr ocytes are estimated to be in the nanomolar concentration range, on th e basis of published values for the activities of glyoxalase I and gly oxalase II in lysed erythrocytes and the steady-state rate of formatio n of D-lactate in intact erythrocytes. This is consistent with a model of the glyoxalase pathway in which the enzyme-catalyzed steps are sig nificantly diffusion limited under physiological conditions.