Some kinetic characteristics of immobilized protomers and native dimers of mitochondrial malate dehydrogenase: an examination of the enzyme mechanism.
Some kinetic characteristics of immobilized native mitochondrial malate dehydrogenase dimers and immobilized protomers, prepared by direct immobilization under conditions yielding complete dissociation without substantial unfolding, were compared to those of native soluble enzyme. Enzyme was covalently immobilized to derivatized porous glass by using a technique which permitted subsequent release of bound enzyme with 0.2 M hydroxylamine at room temperature and pH 7. Kinetic properties of enzyme released from both immobilized dimers and protomers were the same as those for native soluble enzyme, indicating that the immobilization reaction per se did not affect the structure. Both immobilized native dimers and the immobilized protomers exhibited activity with a pH dependence similar to that of native soluble enzyme. The effects of diffusional inhibition were demonstrated for both forms of the immobilized enzyme, especially for the NADH----NAD+ reaction direction. Intrinsic Michaelis constants of both immobilized forms, obtained by extrapolation of apparent values, were similar to those of the soluble enzyme. Furthermore, the effects of inhibitors and effectors with the immobilized forms were the same as those with native soluble enzyme. For example, substrate inhibition was observed with oxalacetate, the inhibitor hydroxymalonate was competitive with ketomalonate and uncompetitive with L-malate, and inhibition was observed with citrate in the NADH----NAD+ direction. Thus, immobilization did not appear to suppress the conformational equilibria of either protomers or dimers. More significantly, the kinetic characteristics of the immobilized protomer were indistinguishable from those of the dimer. Hence, a reciprocating mechanism involving subunit interactions cannot be invoked to explain the allosteric behavior of this dimeric enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013552:Swine; D013646:Tartronates / Q000494:pharmacology
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D013646:Tartronates; D009243:NAD; C032973:tartronic acid; D008291:Malate Dehydrogenase