title: Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate.
abstract: The kinetics of alternative oxidase (AOX) of Arum italicum spadices and soybean (Glycine max L.) cotyledons were studied both with intact mitochondria and with a solubilized, partially purified enzyme. Ubiquinone analogs were screened for their suitability as substrates and ubiquinol-1 was found to be most suitable. The kinetics of ubiquinol-1 oxidation via AOX in both systems followed Michaelis-Menten kinetics, suggesting that the reaction is limited by a single-step substrate reaction. The kinetics are quite different from those previously described, in which the redox state of ubiquinone-10 was monitored and an increase in substrate was accompanied by a decrease in product. The difference between the systems is discussed. Pyruvate is a potent activator of the enzyme and its presence is essential for maximum activity. The addition of pyruvate to the solubilized enzyme increased the maximum initial velocity from 6.2 [plus or minus] 1.3 to 16.9 [plus or minus] 2.8 [mu]mol O2 mg-1 protein min-1 but had little effect on the Michaelis constant for ubiquinol-1, an analog of ubiquinol, which changed from 116 [plus or minus] 73 to 157 [plus or minus] 68 [mu]M. It is concluded that pyruvate (and presumably other keto acids) increases the activity of AOX but does not increase its affinity for its substrate. In agreement with this is the finding that removal of pyruvate (using lactate dehydrogenase and NADH) leads to an 80 to 90% decrease in the reaction rate, suggesting that pyruvate is important in the mechanism of reaction of AOX. The removal of pyruvate from the enzyme required turnover, suggesting that pyruvate is bound to the enzyme and is released during turnover.
authors.affiliation: Department of Botany, University of Adelaide, Adelaide, SA 5005, Australia (M.H.N.H., Q.Z., J.T.W.).
authors.forename: MHN.
authors.initials: M
authors.lastname: Hoefnagel
authors.forename: P. R.
authors.initials: PR
authors.lastname: Rich
authors.forename: Q.
authors.initials: Q
authors.lastname: Zhang
authors.forename: J. T.
authors.initials: JT
authors.lastname: Wiskich