Energetics of S-Adenosylmethionine Synthetase Catalysis

S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase) catalyzes the only known route of biosynthesis of the primary biological alkylating agent. The internal thermodynamics of the Escherichia coli S-adenosylmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphate (PPi), and phosphate (Pi) from ATP, methionine, and water have been determined by a combination of pre-steady-state kinetics, solvent isotope incorporation, 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 tripolyphosphate (PPPi) hydrolysis], and product release. The data demonstrate the presence of a kinetically significant isomerization of the E·AdoMet·PPi·Pi complex before product release. The free energy profile for the enzyme-catalyzed reaction under physiological conditions has been constructed using these experimental values and in vivo concentrations of substrates and products. The free energy profile reveals that the AdoMet formation reaction, which has an equilibrium constant of 104, does not have well-balanced transition state and ground state energies. In contrast, the subsequent PPPi hydrolytic reaction is energetically better balanced. The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet formation and the necessity for subsequent PPPi hydrolysis to allow enzyme turnover. Crystallographic studies have shown that a mobile protein loop gates access to the active site. The present kinetic studies indicate that this loop movement is rapid with respect to k cat and with respect to substrate binding at physiological concentrations. The uniformly slow binding rates of 104−105 M-1 s-1 for ligands with different structures suggest that loop movement may be an intrinsic property of the protein rather than being ligand induced.