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-[alpha] -glycerophosphate dehydrogenase from rabbit muscle, at pH 7.8 and 9.0 at 26 [degree]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 boudn to the enzyme before the addition of the glycerophosphate (L[alpha]GP) or dihydroxyacetone phosphate (DHAP) respectively. At high concentrations NADH, DHAP, and LaGP, 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 LoiGP-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 2 NAD+-binding sites/mole of enzyme is proposed. Alterations of the UV 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 L[alpha]GP caused no alteration in the enzyme spectrum. Spectral changes compatible with the formation of ternary and dead-end complexes were also detected.