Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes.
Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Guha
M K
MK
Department of Chemistry, University of Maryland Baltimore County, Catonsville 21228.
Vander Jagt
D L
DL
Creighton
D J
DJ
D000818:Animals; D002021:Buffers; D004058:Diffusion; D004912:Erythrocytes / Q000201:enzymology*; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008433:Mathematics; D051381:Rats; D013869:Thiolester Hydrolases / Q000097:blood*; D014783:Viscosity; D000099301:Hydroxyacylglutathione Hydrolase
D002021:Buffers; D007202:Indicators and Reagents; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase