Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures

Abstract

31P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, E.CoATP.arginine <=> E.CoADP.P-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289-293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)-31P (gamma-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)-31P distance of approximately 3.0 A, representing direct coordination of Co(II) to gamma-P(ATP), changes to approximately 4.0 A when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)-31P vector implies an excursion of at least 1.0 A for the itinerant phosphoryl group on the surface of the enzyme.