Purification and characterisation of glyoxalase II from human red blood cells.
Glyoxalase II was purified from human red blood cells. The purification factor was 83,300 and the yield was 24% or 1.7 micrograms/ml red blood cells. The purified protein was a monomer with a molecular mass of 29,200 Da and an isoelectric point of 8.3. The rate of hydrolysis of S-D-lactoylglutathione to reduced glutathione and D-lactate, catalysed by glyoxalase II, followed Michaelis-Menten kinetics where the Km and kcat values were 146 +/- 9 microM and 727 +/- 16 s-1, respectively in 50 mM Tris/HCl, pH 7.4 at 37 degrees C. Other S-2-hydroxyacylglutathione derivatives were also acceptable substrates. S-p-Nitrobenzoxycarbonylglutathione was a potent competitive inhibitor of glyoxalase II with a Ki value of 1.20 +/- 0.21 microM, and the hemithioacetal formed non-enzymically from the reaction of methylglyoxal with reduced glutathione was a weak competitive inhibitor with a Ki value of 834 +/- 98 microM.
Allen
R E
RE
Department of Chemistry and Biological Chemistry, University of Essex, Colchester, England.
Lo
T W
TW
Thornalley
P J
PJ
D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification*; D000099301:Hydroxyacylglutathione Hydrolase
D005978:Glutathione; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase