Investigation of the anticooperative binding of NADH to porcine heart mitochondrial malate dehydrogenase.

Affinity labeling of a lysine residue in the coenzyme binding site of pig heart mitochondrial malate dehydrogenase

“…Both NADH and NAD decrease markedly the inactivation rate constant, and the enzyme-coenzyme dissociation constants calculated from the coenzyme concentration dependence of the inactivation rates are comparable to those reported previously on the basis of direct binding measurements (Hodges et al, 1978;Holbrook & Wolfe, 1972). In contrast to the coenzymes, L-malate does not offer marked protection against the inactivation.…”

“…The interaction between the two subunits of malate dehydrogenase has been the focus of considerable speculation, being variously proposed as being responsible for negative cooperativity in coenzyme binding (Bleile et al, 1977;Frieden et al, 1978;Hodges et al, 1978) and for a reciprocating mechanism ("flip-flop") of enzyme catalysis (Harada & Wolfe, 1968). The linear relationship shown here between the stoichiometry of modification of malate dehydrogenase by 5'-[/>-(fluorosulfonyl)benzoyl] adenosine and loss of catalytic activity suggests that the subunits may not be obligatorily coupled in the catalytic cycle.…”