Halibut muscle 3-phosphoglycerate kinase. Chemical and physical properties of the enzyme and its substrate complexes.
An efficient procedure for the purification of 3-phosphoglycerate kinase (PGK) from Pacific halibut muscle is described. The molecular weight (43500) and specific activity are similar to those of other species of PGK. The isoelectric point (greater than 9.5) is more than 1.4 pH units higher than that reported for mammalian muscle PGK. The reaction of the seven thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is kinetically biphasic; reaction at a single fast-reacting thiol inactivates the enzyme. The binding of all substrates and products to PGK was observed by 31P NMR. 1,3-Diphosphoglycerate (1,3-P2G) is more tightly bound than is any of the other reaction components. Unlike 1,3-P2G in aqueous solution, the complex with PGK is protected from hydrolysis over a period of weeks. The 31P chemical shifts of this complex are insensitive to pH which suggests that solvent water is excluded from the substrate-bound cleft. As with yeast PGK, the equilibrium constant for the phosphoryl transfer reaction is near unity in the enzyme site environment in contrast to a value of approximately 10(3) (in favor of ATP) in aqueous solution. Since the ternary complex equilibrium 31P NMR spectrum can be accounted for entirely on the basis of the various binary complex spectra, there is no compelling evidence for the involvement of a stoichiometrically substantial phosphoenzyme intermediate.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005399:Fishes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology*; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013438:Sulfhydryl Compounds
D004163:Diphosphoglyceric Acids; D013438:Sulfhydryl Compounds; C015891:glycerate 1,3-biphosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D010735:Phosphoglycerate Kinase