Characterization of two members of a novel malic enzyme class.
The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.
Voegele
R T
RT
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ont. L8S 4K1, Canada.
Mitsch
M J
MJ
Finan
T M
TM
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D003001:Cloning, Molecular; D003653:Decarboxylation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005650:Fumarates / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016962:Sinorhizobium meliloti / Q000235:genetics; D019802:Succinic Acid / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005650:Fumarates; D007527:Isoenzymes; D000105:Acetyl Coenzyme A; C032005:fumaric acid; D019802:Succinic Acid; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)