ROLE OF METAL COFACTORS IN ENZYME REGULATION - DIFFERENCES IN THE REGULATORY PROPERTIES OF THE ESCHERICHIA-COLI NICOTINAMIDE ADENINE-DINUCLEOTIDE SPECIFIC MALIC ENZYME DEPENDING ON WHETHER MG2+ OR MN2+ SERVES AS DIVALENT-CATION

A number of differences in the kinetic properties of the E. coli NAD+ dependent malic enzyme were found depending on whether Mg2+ or Mn2+ served to fulfill the divalent cation requirement. With Mg2+ as cation, the velocity-malate saturation curve in the absence of effectors is complex at pH 7.4, indicating a combination of apparent positive and negative cooperativity, while the velocity-free Mg2+ saturation curve exhibits positive cooperativity. If Mn2+ serves as cation, the velocity-malate and velocity-free Mn2+ saturation curves exhibit a simple hyperbolic response. The velocity-NAD+ saturation curves, in contrast, exhibit a simple hyperbolic response in the presence of either metal cofactor, but the affinity for NAD+ and the Vmax are increased in the presence of Mn2+. When Mg2+ serves as cation, the enzyme activity is much more sensitive to regulation by the allosteric inhibitor CoA and the allosteric activator aspartate. If Mn2+ replaces Mg2+, the enzyme activity is more sensitive to inhibition by ATP. This inhibition is due to chelation but may be of physiological importance. The inhibitor, CoA, increases the interaction between malate-binding sites in the presence of Mn2+ but has little effect on subunit interaction in the presence of Mg2+. The kinetic data can be explained by a model involving sequential ligand-induced conformational changes of the enzyme, resulting in a mixture of apparent positive and negative cooperative behavior. Alternate explanations involving different classes of noninteracting binding sites or different enzyme forms are also considered. The metal cofactors Mg2+ and Mn2+ appear to stabilize 2 distinct forms of the enzyme which differ in response to varying substrate and effector concentrations. The results are strikingly similar to previous results reported on the NAD+-dependent isocitrate dehydrogenase, supporting the suggestion that metal cofactors function as regulatory entities.