Metabolism of 2-oxoaldehyde in yeasts. Purification and characterization of NADPH-dependent methylglyoxal-reducing enzyme from Saccharomyces cerevisiae.
An enzyme catalyzing the reduction of methylglyoxal was isolated from Saccharomyces cerevisiae and its enzymatic properties were analyzed. The enzyme, specifically eluted from a blue-dextran--Sepharose CL-6B column by the substrate, methylglyoxal, was homogeneous on polyacrylamide gel electrophoresis. The enzyme consisted of single polypeptide chain with a relative molecular mass of 43 000. The enzyme was glycoprotein and contained 6.6% carbohydrate. NADPH was specifically required for activity and the Km for NADPH was 2.0 X 10(-7) M. The enzyme was active on various glyoxals such as glyoxal, methylglyoxal (Km = 5.88 mM) and phenylglyoxal (Km = 1.54 mM). The reaction catalyzed by the enzyme was virtually irreversible. The activity was inhibited by sulfhydryl agents and activated by reducing agents such as glutathione. Intermediates in glycolysis, nucleosides, nucleotides, polyamines and various metal ions showed little inhibitory or activating effects on enzyme activity. Tricarboxylic acids showed a slight inhibitory effect. The activity of the enzyme was strongly inhibited by anionic detergents. The enzyme was rapidly inactivated by incubating with the substrates probably because of the non-enzymatic interaction between glyoxals and NH2 groups in arginine residues in the enzyme. NADP, one of the reaction products, also inhibited the enzyme activity and the Ki for NADP was about 0.07 mM. We tentatively designated the enzyme methylglyoxal reductase.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D002241:Carbohydrates; D009249:NADP; D000429:Alcohol Oxidoreductases; C031416:D-lactaldehyde dehydrogenase