ICAR Research Data Repository for Knowledge Management

Rate studies using phosphoglycerate
kinase (PGK) - glyceraldehyde-3-phosphate dehydrogenase (GPDH) enzyme pair have
been carried out to distinguish between the two mechanisms of intermediate
metabolite transfer, namely diffusion through the solvent versus "substrate
channelling" within an enzyme-enzyme complex. A procedure has been
described for the assay of the rates of PGK-catalysed and the PGK-GPDH coupled
reactions at high (saturating) GPDH concentration. 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 KCI), where a
PGK-GPDH complex is known to dissociate, the two reactions proceeded at almost equal
rates. At fixed PGK concentration, the rate of the coupled reaction at high
(saturating) GPDH concentration varied with the nature (biological origin) of
the latter enzyme. In the presence of 0.5 M KCI, the saturating rate
values with different GPDHs were almost equal. The PGK-catalysed reaction
exhibited typical Michaelian behaviour on varying the substrate concentrations
(linear double reciprocal plots). The K_m values for 3-PGA (0.51 mM) and ATP (0.40 rnM) were independent of the concentration of the second substrate.
The double reciprocal plots for the coupled reaction showed downward curvature,
i.e. activation at higher substrate concentrations. The ratio of the rate of
the coupled reaction : the rate of the PGK catalysed reaction was found to be a
function of the nature of PGK, nature of GPDH, nature of buffer, pH, salt concentration and
substrate concentrations. The ratio varied between close to unity at low
substrate concentrations, to three when the V _max
values of the two reactions were compared. At low substrate concentrations, the
rate of the coupled reaction became independent of the nature of GPDH. It has
been suggested that in the PGK-GPDH pair, the intermediate metabolite (BPG) is
transferred directly from one enzyme to the other within an enzyme-enzyme
complex, except at high salt or low substrate concentrations. Under the latter
conditions, data were consistent with metabolite transfer by diffusion.
Implications of these results for coupled enzyme assays have been discussed.