The N-ethylmaleimide-sensitive cysteine residue in the pH-dependent subunit interactions of malate dehydrogenase.
The specific chemical modification by N-ethylmaleimide of a cysteine residue at pH 5.0 in porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been shown to result in an enzymatically inactive, monomeric product, which does not reassociate at pH 7.5 to yield the native dimer. In this report, an investigation of proton release and uptake upon NADH binding to the native enzyme and to the N-ethylmaleimide-modified enzyme has implicated the above cysteine residue as being directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase. The results are consistent with the view that the modified cysteine residue is not located at the subunit interaction site, although it is probably near this site. A recent study from this laboratory has demonstrated that the monomeric enzyme obtained at pH 5.0 exists in a conformation which is enzymatically inactive and which has an enhanced intrinsic protein fluorescence. Interpretation of protein fluorescence data has suggested that the N-ethylmaleimide modification results in inactivation of the enzyme by preventing the pH-induced conformational change to the active dimer. However, NADH is able to induce reassociation of the N-ethylmaleimide-modified enzyme at pH 7.5 but not at pH 5.0. This reassociation at pH 7.5 is accompanied by a significant regain of enzymatic activity, indicating that NADH binding is able to partially overcome the negative effect of the cysteine modification on the pH-dependent subunit reassociation of mitochondrial malate dehydrogenase.
D000818:Animals; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD; D010084:Oxidation-Reduction; D013552:Swine
D046911:Macromolecular Substances; D009243:NAD; D008291:Malate Dehydrogenase; D003545:Cysteine; D005033:Ethylmaleimide