Kinetics of carbonyl reductase from human brain.
Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. Deuterium isotope effects on the apparent V/Km values decreased with increasing concentrations of menadione but were independent of the NADPH concentration. The results, together with data from product inhibition studies, are consistent with carbonyl reductase obeying a compulsory-order mechanism, NADPH binding first and NADP+ leaving last. No significant differences in the kinetic properties of three molecular forms of carbonyl reductase were detectable.
Bohren
K M
KM
Institut für Biochemie und Molekularbiologie der Universität Bern, Switzerland.
von Wartburg
J P
JP
Wermuth
B
B
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D003903:Deuterium / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D014812:Vitamin K / Q000378:metabolism
D014812:Vitamin K; D009249:NADP; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)