Essential sulfhydryl for reduced nicotinamide adenine dinucleotide binding in D-.beta.-hydroxybutyrate dehydrogenase

Abstract

Chemical derivatization studies were directed at the SH group of [bovine heart mitochondria] D-.beta.-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme. Reaction with N-ethylmaleimide (NEM) leads to progressive and parallel loss of both enzymic activity and coenzyme binding. Both functions are lost when 1 equivalent of SH is derivatized/mol of enzyme. Inactivation of the enzyme with methylmercury or with air oxidation also leads to loss of coenzyme binding. A single essential SH is probably required for coenzyme binding and consequently for enzymic activity. Only 2 accessible cysteine residues can be derivatized even at high levels of NEM, whereas derivatization of the remaining 3 inaccessible cysteines requires denaturation of the enzyme. The enzyme can apparently be labeled in the accessible, but nonessential, SH in the presence of coenzyme which protects against inactivation by NEM. Such selective covalent labeling of the nonessential SH makes possible future biophysical studies of enzyme-phospholipid interaction of a functional enzyme using extrinsic probes.