KINETIC STUDIES ON THE MECHANISM OF CYTOPLASMIC L-α-GLYCEROPHOSPHATE DEHYDROGENASE OF RABBIT SKELETAL MUSCLE

Abstract

Studies on initial velocity and product inhibition were carried out on crystalline cytoplasmic NAD+-linked L-α-glycerophosphate dehydrogenase from rabbit muscle, at pH 7.8 and 9.0 at 26 °C. Michaelis and inhibition constants for all the reactants were determined. The kinetic data were consistent with an ordered mechanism in which nicotinamide–adenine dinucleotide (NAD+) or its reduced form (NADH) is bound to the enzyme before the addition of the glycerophosphate (LαGP) or dihydroxyacetone phosphate (DHAP) respectively. At high concentrations NADH, DHAP, and LαGP, but not NAD+, produced substrate inhibition. Combined product-inhibition and dead-end inhibition studies indicated the formation of inactive dead-end complexes of NADH–enzyme, DHAP–enzyme, and LαGP–enzyme–NADH. The low rate constant calculated for the dissociation of the active NADH–enzyme complex suggested an ordered mechanism involving either the formation of an inactive dead-end NADH–enzyme complex or an isomerized NADH–enzyme complex. A choice between these possibilities could not be made on the basis of the present kinetic data. A mechanism for substrate inhibition involving two NAD+-binding sites per mole of enzyme is proposed. Alterations of the ultraviolet absorption spectrum of the enzyme by NAD+ and NADH were in agreement with the conclusion from the kinetic results that the coenzymes are bound to the enzyme before the substrates. DHAP and LαGP caused no alteration in the enzyme spectrum. Spectral changes compatible with the formation of ternary and dead-end complexes were also detected.