Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.
NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP; D011189:Potassium Chloride / Q000494:pharmacology
D000596:Amino Acids; D009249:NADP; D011189:Potassium Chloride; D000105:Acetyl Coenzyme A; D008291:Malate Dehydrogenase