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)/Km (3.5 x 10(6) M(-1) s(-1), pH 7, 25 degrees 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(cat)/Km for the slow substrate GSH-t-butylglyoxal thiohemiacetal (3.5 x 10(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 about 50% diffusion limited under conditions where [substrate] << Km; 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.
Shih
M J
MJ
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore 21228, USA.
Edinger
J W
JW
Creighton
D J
DJ
D000818:Animals; D004058:Diffusion; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000097:blood / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014783:Viscosity
D007791:Lactoylglutathione Lyase