INVESTIGATION OF RELATION OF PH-DEPENDENT DISSOCIATION OF MALATE-DEHYDROGENASE TO MODIFICATION OF ENZYME BY N-ETHYLMALEIMIDE

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 252  (17) , 6038-6041
Abstract
The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was further characterized using sedimentation velocity ultracentrifugation. The increased rate and specificity of inactivation of mitochondrial malate dehydrogenase by the sulfhydryl reagent N-ethylmaleimide was correlated with the pH-dependent dissociation of the enzyme. Data obtained using NAD+ and its component parts to reassociate the enzyme and to protect the enzyme from inactivation by N-ethylmaleimide suggested that the sulfhydryl residues, being modified by N-ethylmaleimide, were inaccessible when the enzyme was in its dimeric form. A dissociation curve for the pH-dependent dissociation suggested that a limited number of residues were being protonated concomitant with dissociation of the enzyme. An apparent pKa [log of the reciprocal of the acidic dissociation constant] of 5.3 was determined for this phenomenon. Studies using enzyme modified by the sulfhydryl reagent N-ethylmaleimide indicated that selective modification of essential sulfhydryl residues altered proper binding of NADH.

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