Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase

Abstract

Measurements of the relaxation rate of water protons (PRR) were used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. Phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase and arginine kinase, with maximal binding of metal ion to the enzyme in the presence of the nucleotide substrate. An analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately 2 orders of magnitude more tightly than the free nucleotide. Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by EPR studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. The dissociation constant for the enzyme-MnATP complex is approximately 4-fold lower than that for enzyme-ATP, and there are no substantial changes in the EPR spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. Addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II).