Energetics of S-adenosylmethionine synthetase catalysis

S-Adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) ca talyzes the only known route of biosynthesis of the primary biological alky lating agent. The internal thermodynamics of the Escherichia coli S-adenosy lmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphat e (PPi), and phosphate (P-i) from ATP, methionine, and water have been dete rmined by a combination of pre-steady-state kinetics, solvent isotope incor poration, and equilibrium binding measurements in conjunction with computer modeling. These studies provided the rate constants for substrate binding, the two chemical interconversion steps [AdoMet formation and subsequent tr ipolyphosphate (PPPi) hydrolysis], and product release. The data demonstrat e the presence of a kinetically significant isomerization of the E.AdoMet.P Pi.P-i complex before product release. The free energy profile for the enzy me-catalyzed reaction under physiological conditions has been constructed u sing these experimental values and in vivo concentrations of substrates and products. The free energy profile reveals that the AdoMet formation reacti on, which has an equilibrium constant of 10(4), does not have well-balanced transition state and ground state energies. In contrast, the subsequent PP Pi hydrolytic reaction is energetically better balanced, The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet forma tion and the necessity for subsequent PPPi hydrolysis to allow enzyme turno ver. Crystallographic studies have shown that a mobile protein loop gates a ccess to the active site. The present kinetic studies indicate that this lo op movement is rapid with respect to k(cat) and with respect to substrate b inding at physiological concentrations, The uniformly slow binding rates of 10(4)-10(5) M-1 s(-1) for ligands with different structures suggest that l oop movement may be an intrinsic property of the protein rather than being ligand induced.