The Glyceraldehyde 3-Phosphate Dehydrogenases of Liver and Muscle: COOPERATIVE INTERACTIONS AND CONDITIONS FOR FUNCTIONAL REVERSIBILITY

Abstract

Abstract A method is described for the isolation of glyceraldehyde 3-phosphate dehydrogenase from rabbit liver. The enzyme has been crystallized as the NAD complex and its chemical and physical properties have been compared with those of the muscle enzyme. The kinetics of the reversible reaction catalyzed by the dehydrogenases is sensitive to temperature and ionic strength and has been examined at 37°, pH 7.4, in a solvent of ionic strength 0.1. Under these conditions the outstanding new feature of the reverse reaction, the reductive dephosphorylation of 3-phosphoglyceroyl phosphate, is the positive cooperativity of the response of both enzymes to concentration increments of the acyl phosphate and the increase in this cooperativity as a function of concentration of the cosubstrate, NADH. At low concentrations of the acyl phosphate, the reduced coenzyme exerts an inhibitory function which is abolished by high concentrations of acyl phosphate or by low concentrations of NAD acting as a heterotropic effector. The same concentrations of NAD which activate at low acyl phosphate concentration inhibit at high concentrations of the 3-carbon substrate by competing with NADH. Activation of the reaction by NAD involves a transition of the acyl phosphate saturation function from a sigmoidal to the hyperbolic form and hence sensitizes the enzyme to lower concentrations of acyl phosphate. The substrate inhibition by NADH and the activation by NAD may be accounted for by the binding of the nucleotides to catalytic sites of the tetrameric protein that are not occupied by the acyl enzyme intermediate. Although the competitive advantage in binding to the apoprotein strongly favors NAD over NADH, the competition under reaction conditions tends to be equalized at nonacylated sites of the acylated protein and is drastically reversed at the acylated sites. Thus at kinetic saturation levels of the acyl phosphate Knadh remains in the 10-µm range, but half-maximal inhibition by NAD occurs at concentrations of the latter that are in excess of 1000 µm. These properties involve both ligandinduced conformational transitions of the protein and a local steric effect of the acyl group that interferes with the binding of the oxidized but not the reduced form of the pyridine nucleotide. In the forward or oxidative phosphorylation reaction the reciprocal kinetic plots are entirely linear except under conditions of product inhibition by NADH. The preferential binding of NADH at an acylated site is not strongly expressed in the inhibition by added NADH because such sites are already occupied by product NADH that is released subsequent to the rate-limiting acyl group transfer from enzyme to orthophosphate ion. The acyl phosphate is a strong inhibitor of the forward reaction, noncompetitive with NAD and strictly competitive with aldehyde. Intracellularly, the enzyme always operates in the presence of high concentrations of NAD which are required to provide the driving force for the energy-conserving reaction. Functional reversibility, under the adverse substrate concentration distributions in the aerobic hepatocyte, is maintained by regulatory effects of NAD binding which promote the reaction of extremely low concentrations of acyl phosphate, and by the differential effects of acylation of the protein which allow access of substrate NADH and at the same time eliminate a prohibitive inhibition by the high concentrations of NAD. The catalytic properties of the liver and muscle enzyme are qualitatively similar but exhibit some quantitative differences that would favor the more diversified metabolic requirements of liver. However, no definitive structural differences have been identified.