Phosphoglycerate kinase glyceraldehyde-3-phosphate dehydrogenase interaction: Reaction rate studies

studies using phosphoglycerate kinase (PGK)-glyceraldehyde-3-phosphate dehy drogenase (GPDH) enzyme pair have been carried out to distinguish between t he two mechanisms of intermediate metabolite transfer, namely diffusion thr ough the solvent versus "substrate channelling" within an enzyme-enzyme com plex. A procedure has been described for the assay of the rates of PGK-cata lysed and the PGK-GPDH coupled reactions at high (saturating) GPDH concentr ation. With PGKs of rabbit muscle and yeast, the coupled reaction proceeded faster than the PGK-catalysed reaction. At a high salt concentration (0.5 M KCl), where a PGK-GPDH complex is known to dissociate, the two reactions proceeded at almost equal rates. At fixed PGK concentration, the rate of th e coupled reaction at high (saturating) GPDH concentration varied with the nature (biological origin) of the latter enzyme. In the presence of 0.5 M K Cl, the saturating rate values with different GPDHs were almost equal. The PGK-catalysed reaction exhibited typical Michaelian behaviour on varying th e substrate concentrations (linear double reciprocal plots). The K-m values for 3-PGA (0.51 mM) and ATP (0.40 mM) were independent of the concentratio n of the second substrate. The double reciprocal plots for the coupled reac tion showed downward curvature, i.e. activation at higher substrate concent rations. The ratio of the rate of the coupled reaction : the rate of the PG K catalysed reaction was found to be a function of the nature of PGK, natur e of GPDH, nature of buffer, pH, salt concentration and substrate concentra tions. The ratio varied between close to unity at low substrate concentrati ons, to three when the V-max values of the two reactions were compared. At low substrate concentrations, the rate of the coupled reaction became indep endent of the nature of GPDH. It has been suggested that in the PGK-GPDH pa ir, the intermediate metabolite (BPG) is transferred directly from one enzy me to the other within an enzyme-enzyme complex, except at high salt or low substrate concentrations. Under the latter conditions, data were consisten t with metabolite transfer by diffusion. Implications of these results for coupled enzyme assays have been discussed.