Pyruvate:NADP + oxidoreductase from Euglena gracilis: The kinetic properties of the enzyme

Abstract

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 K m values for pyruvate, CoA, and NADP + were estimated to be 27, 6.6, and 28 μ m, respectively, and the K i value of CoA against pyruvate was 28 μ m. CO 2 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 CO 2 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.