P. Prabhakar et al., Phosphoglycerate kinase glyceraldehyde-3-phosphate dehydrogenase interaction: Reaction rate studies, I J BIOCH B, 36(2), 1999, pp. 88-100
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.