The purification and properties of Escherichia coli methylglyoxal synthase.
1. Methylglyoxal synthase was purified over 1500-fold from glycerol-grown Escherichia coli K 12 strain CA 244. The purified enzyme was inactivated by heat or proteolysis, had a molecular weight of approx. 67000, a pH optimum of 7.5 and was specific for dihydroxyacetone phosphate with K(m) 0.47mm. 2. The possibility that a Schiff-base intermediate was involved in the reaction mechanism was investigated but not confirmed. 3. The purified enzyme lost activity, especially at low temperature, but could be stabilized by P(i). Two binding sites for P(i) may be present on the enzyme. Of other compounds tested only the substrate, dihydroxyacetone phosphate, and bovine serum albumin showed any significant stabilizing effect. 4. Phosphoenolpyruvate, 3-phosphoglycerate, PP(i) and P(i) were potent inhibitors of the enzyme. Kinetic experiments showed that PP(i) was apparently a simple competitive inhibitor, but inhibition by the other compounds was more complex. In the presence of P(i) the enzyme behaved co-operatively, with at least three binding sites for dihydroxyacetone phosphate. 5. It is proposed that methylglyoxal synthase and glyceraldehyde 3-phosphate dehydrogenase play important roles in the catabolism of the triose phosphates in E. coli. Channelling of dihydroxyacetone phosphate via methylglyoxal would not be linked to ATP formation and could be involved in the uncoupling of catabolism and anabolism.
D000096:Acetone; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008190:Lyases / Q000302:isolation & purification*; D008970:Molecular Weight; D009943:Organophosphorus Compounds; D012710:Serum Albumin, Bovine; D014306:Trioses
D009943:Organophosphorus Compounds; D014306:Trioses; D000096:Acetone; D012710:Serum Albumin, Bovine; D000255:Adenosine Triphosphate; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C021578:methylglyoxal synthase