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

Abstract

EVANS, C. T., and C. RATLEDGE. 1985. Partial purification and properties of pyruvate kinase and its regulatory role during lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14. Can. J. Microbiol. 31: 479-484. 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 (M,) = 190000) showed a pH optimum of 8.0 and apparent Km values for K', phosphoenolpyruvate (PEP), and ADP of 1.6 mM, 571 pM, and 120 pM, respectively. Enzyme activity was inhibited by citrate, isocitrate, ATP, GTP, and CTP and activated by fructose 1,6-bisphosphate, L-glutamate, and NH: ions. Inhibition by citrate and ATP were both competitive with PEP with the Kicciiraie, = 340 pM and Ki(ATp) = 303 pM. 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 I,6-bisphosphate increased the affinity of the enzyme for both PEP and ADP and so were significant activators at nonsaturating substrate concentrations. N H~ 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.

Mitochondrial Efflux of Citrate and Isocitrate Is Fully Dispensable for Glucose-Stimulated Insulin Secretion and Pancreatic Islet β-Cell Function

“…On the basis of these data, we propose that Idh1 functions to relieve citrate/ isocitrate-mediated inhibition of a glycolytic enzyme to maintain steady rates of glucose metabolic flux rather than Idh1 acting as a direct positive regulator of GSIS as previously suggested (16). Citrate is well established to allosterically inhibit phosphofructokinase-1 (42,43), the rate-limiting step in glycolysis (particularly in the context of increased ATP levels) as well as pyruvate kinase (44,45), which are critical to carbon flow into the mitochondria. Considering that the essential function of the islet b-cell is to rapidly coordinate dose-dependent changes in blood glucose levels with insulin secretion, limiting substrate-dependent allosteric inhibition of glycolysis at two key regulatory nodes would seem to be a critical evolutionary advantage to ensure b-cell responsivity within the full range of physiological glucose levels.…”

Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate

“…Citrate is an important allosteric effector of several enzymes involved in fundamental pathways of cellular metabolism, such as acetyl-CoA carboxylase (Gregolin et al, 1968a,b), pyruvate kinase (Miyanaga et al, 1984;Chrispeels & Gaede, 1985;Storey, 1985;Evans & Ratledge, 1985) and phosphofructokinase (Passonneau & Lowry, 1963;Foe & Kemp, 1982;Narabayashi et al, 1985). With reference to the tricarboxylic acid cycle, citrate activates isocitrate dehydrogenase (Gabriel & Plaut, 1984) and inhibits fumarase (Cennamo et al, 1967).…”

Structural and kinetic characterization of Trypanosoma congolense pyruvate kinase

“…The asymmetric unit of the crystal The presence of citrate in TcoPYK's active site could also imply that citrate potentially inhibits its activity through locking the enzyme in a non-productive R-state. Indeed, citrate has been reported to inhibit PYKs from various organ- isms; Plasmodium falciparum [43], cyanobacterium Synechococcus [44], Solanum tuberosum (potato) [45], Glycine max (soybean) [46], yeast Rhodosporidium toruloides [47], HsaLPYK [42] and Leishmania major PYK [48]. In the latter case, the addition of 10 mM citrate leads to a reduction of 50% in enzyme activity.…”