Dihydroxyacetone kinase of methanol-assimilating yeast. I. Regulation of dihydroxyacetone kinase from Candida methylica in situ.

Abstract

In order to investigate the control behaviour of dihydroxyacetone kinase of methanol-grown Candida methylica under nearly physiological conditions kinetic and regulatory studies were carried out in situ. Yeast cells were made permeable to substrate and reaction products by treatment with Triton X-100. Normal Michaelis-Menten kinetics resulted in dependence upon the dihydroxyacetone concentration, both at the pH optimum of 7.6 and near the physiological pH-value of 6.5. The Km obtained for dihydroxyacetone was 0.02 mM, independent of the pH-value. The plots of dihydroxyacetone kinase activity as a function of the ATP concentration revealed complex kinetic characteristics with plateau regions. The maximum reaction rate was reached only after a lag time both at pH 7.6 and concentrations of ATP higher than 5 mM and at pH 6.5 and concentrations of ATP higher than 1.25 mM. Among a great number of tested metabolites no inhibitors of dihydroxyacetone kinase were found. Dihydroxyacetone kinase activity depending upon energy charge according to Atkinson exhibited curves of the U-type. These results and further data concerning the regulation of other enzymes obtained with C. methylica and other yeasts were the basis to propose a preliminary overall model of fine control of the carbon and energy metabolism of methanol-utilizing yeasts.