Complex of brain d-phosphoglycerate mutase and γ enolase and its reactivation by d-glycerate 2,3-bisphosphate

Abstract

The dissociabilities of dimeric γ enolase, α enolase, and phosphoglycerate mutase of brain origin were tested using fluorescein isothiocyanate attached covalently to these enzymes. The dissociation constant of dimeric γ enolase is lower ( K d = 0.03 μM) than that of the α enolase ( K d = 3 μM), while dimeric mutase seems to be nondissociable in the concentration range 0.1–10 μ m, at pH 7.3 in 50 m m imidazole buffer at 20 °C. Interaction of neuron-specific γ enolase with d-phosphoglycerate mutase was detected with the same fluorescence-labeling technique as well as by a kinetic analysis. The determined dissociation constant of the enolase-mutase complex was found to be in the range 5–40 μ m, independent of the technique used. A mixed type of inhibition in the binding of d-glycerate-2-P and mutase to the d-glycerate-2-P binding site on enolase was observed in the absence of d-glycerate-2,3-P 2. However, the inhibition of the enolase activity by brain d-phosphoglycerate mutase in the d-glycerate-2-P → phospho enolpyruvate transformation is almost fully reverted by d-glycerate-2,3-P 2, probably via the proper coordination of the active centers in the ternary complex of enolase, d-phosphoglycerate mutase, and their common intermediate, d-glycerate-2-P. The mechanism of intermediate transfer by consecutive enzyme pairs in a nondivergent metabolite flux (around the transformation of d-glycerate-2-P) is examined and conclusions of the present experiments are compared with the results of an extended analysis performed earlier with a divergent metabolite flux (around the transformation of multiusage triosephosphates, d-glyceraldehyde-3-P, and dihydroxyacetone phosphate).