Partial purification and properties of pyruvate kinase and its regulatory role during lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14

Abstract

Pyruvate kinase from the oleaginous yeast Rhodosporidium toruloides CBS 14 was partially purified and its properties investigated to determine its role during lipid production by this yeast. The enzyme (relative mass (Mr) = 190 000) showed a pH optimum of 8.0 and apparent Km values for K+, phosphoenolpyruvate (PEP), and ADP of 1.6 mM, 571 μM, and 120 μM, respectively. Enzyme activity was inhibited by citrate, isocitrate, ATP, GTP, and CTP and activated by fructose 1,6-bisphosphate, L-glutamate, and [Formula: see text] ions. Inhibition by citrate and ATP were both competitive with PEP with the Ki(citrate) = 340 μM and Ki(ATP) = 303 μM. The effect of ATP and cellular energy charge were critically dependent on the concentration of ADP present in the enzyme assay. Both L-glutamate and fructose 1,6-bisphosphate increased the affinity of the enzyme for both PEP and ADP and so were significant activators at nonsaturating substrate concentrations. [Formula: see text] ions increased the affinity of the enzyme for PEP, but not ADP. The modulation of pyruvate kinase activity by such a wide range of effectors is indicative of a major regulatory role in controlling the flux of carbon, through glycolysis, into lipid-synthesizing systems.