Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme.
The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.
Inui
H
H
Department of Food and Nutrition, Narabunka Women's Junior College, Japan.
Miyatake
K
K
Nakano
Y
Y
Kitaoka
S
S
D000818:Animals; D001667:Binding, Competitive; D003065:Coenzyme A / Q000378:metabolism; D003653:Decarboxylation; D005056:Euglena gracilis / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010269:Paraquat; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013379:Substrate Specificity
D011773:Pyruvates; D009249:NADP; D019289:Pyruvic Acid; D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D010269:Paraquat; D003065:Coenzyme A