J. Biol. Chem., Vol. 256, Issue 19, 9895-9900, Oct, 1981
The N-ethylmaleimide-sensitive cysteine residue in the pH-dependent subunit interactions of malate dehydrogenase
DC Wood, CT Hodges, SM Howell, LG Clary and JH Harrison
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.
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