Complex of brain D-phosphoglycerate mutase and gamma enolase and its reactivation by D-glycerate 2,3-bisphosphate.
The dissociabilities of dimeric gamma enolase, alpha enolase, and phosphoglycerate mutase of brain origin were tested using fluorescein isothiocyanate attached covalently to these enzymes. The dissociation constant of dimeric gamma enolase is lower (Kd = 0.03 microM) than that of the alpha enolase (Kd = 3 microM), while dimeric mutase seems to be nondissociable in the concentration range 0.1-10 microM, at pH 7.3 in 50 mM imidazole buffer at 20 degrees C. Interaction of neuron-specific gamma 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 microM, 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-P2. However, the inhibition of the enolase activity by brain D-phosphoglycerate mutase in the D-glycerate-2-P----phosphoenolpyruvate transformation is almost fully reverted by D-glycerate-2,3-P2, 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).
Batke
J
J
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest.
Nazaryan
K B
KB
Karapetian
N H
NH
D019794:2,3-Diphosphoglycerate; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004163:Diphosphoglyceric Acids / Q000494:pharmacology*; D004793:Enzyme Reactivators; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D010736:Phosphoglycerate Mutase / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D004163:Diphosphoglyceric Acids; D004793:Enzyme Reactivators; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013861:Thiocyanates; D019794:2,3-Diphosphoglycerate; D010770:Phosphotransferases; D010751:Phosphopyruvate Hydratase; D010736:Phosphoglycerate Mutase; D016650:Fluorescein-5-isothiocyanate