[Properties of pyruvate carboxylase of the facultative methylotrope Pseudomonas oleovorans].

Pyruvate carboxylase of the facultative methylotroph Pseudomonas oleovorans was purified 40-fold by ammonium sulfate fractionation, gel filtration on Ultrogel AcA 34, ion-exchange chromatography on DEAE-Biogel A and concentration on DEAE-Sepharose CL-6B. The enzyme exerts its maximal activity in the presence of Mg2+ (pH 7.5, 40 degrees), is unstable and completely inactivated within 6 hrs at 25 degrees. In the presence of Mg2+ monovalent cations stimulate the enzyme activity. The molecular weight of pyruvate carboxylase as determined by gel filtration of Sepharose CL-6B is 300,000. The enzyme molecule contains biotin. The apparent Km values for pyruvate, ATP and HCO3- are 1.77, 0.19 and 0.23 mM, respectively. CoASAc, alpha-ketoglutarate and glutamate have no effect on the enzyme activity. The enzyme is inhibited by aspartate, malate, oxaloacetate and is activated by citrate, isocitrate and phosphosugars. The role of pyruvate carboxylase in methylotrophic metabolism of Ps. oleovorans is discussed.