Kinetic and magnetic resonance studies of the pyruvate kinase reaction. II. Complexes of enzyme, metal, and substrates.

Abstract A correlation between the kinetics of the pyruvate kinase reaction and the enhancements of the relaxation rate of the nuclear spins of water protons in the ternary enzyme-manganese-substrate (E-Mn-S) complexes, et, has revealed certain salient features of the pathway of the reaction and the structure of the ternary intermediates. The rate equation for initial velocities of the forward reaction was derived by assuming (a) rapid simultaneous equilibria of enzyme with divalent metal ion, adenosine diphosphate, manganese adenosine diphosphate, and phosphoenolpyruvate, (b) random order of combination, and (c) phosphoryl transfer as the rate-determining step. This formulation permitted the calculation of the dissociation constants Ks of the ES complexes and K3 of the EMS complexes for ADP and phosphoenolpyruvate. The values of K3 for E-Mn-ADP and E-Mn-phosphoenolpyruvate were determined directly by equilibrium experiments with proton relaxation rate (PRR) with the use of et as the parameter which characterized the ternary complexes. The values of Ks for E-phosphoenolpyruvate and E-ADP were determined directly by equilibrium experiments with the use of the kinetic protection method with p-chloromercuribenzoate inactivation. The agreement of the dissociation constants of the binary and ternary enzyme complexes determined by equilibrium methods with the kinetically calculated ones, taken in conjunction with the previously established agreement of the dissociation constant of EM with its kinetically calculated activator constant, validates the assumed random binding of metal, ADP, MnADP, and phosphoenolpyruvate to the enzyme. The inhibitor constant, Ki, for ATP agreed well with its K3 determined by PRR. In the ternary pyruvate complex, the results from PRR data differed from the kinetic data in two ways: pyruvate was not competitive with phosphoenol pyruvate, and K3 from PRR data was an order of magnitude lower than Ki obtained kinetically. The value of K3 was raised to the Ki value when the PRR titration was performed in the presence of ADP or inorganic phosphate, suggesting that the breakdown of the product complex in the pyruvate kinase reaction proceeds by a preferred order in which pyruvate dissociates first. The PRR enhancement values of all the ternary EMS complexes, et, were found to be less than eb, the enhancement of the binary complex; this decrease may be ascribed in part to the replacement of water ligands by groups on the substrate. The magnitude of the decrease of et(ATP) with respect to et(ADP) is consistent with a metal bridge structure in which the divalent cation is coordinated both to the enzyme and to the γ-phosphoryl group of ATP in the active complex. No data have been found which contravene a metal bridge structure. Kinetic data which favor this structure may be adduced from the relative binding constants of substrates to the manganese- and magnesium-activated enzyme; changing the divalent activator from manganese to magnesium, which has a lower affinity for ligands, raised the K3 of phosphoenolpyruvate and the Ki of ATP and of pyruvate by factors of 2.6, 5.1, and 2.6, respectively, but it had little effect on the K3 of ADP. The structure of the E-Mn-phosphoenolpyruvate complex as reflected in the finding that et(phosphoenolpyruvate) << eb requires a change in the environment of the manganese other than mere replacement of water ligands; such a change may be ascribed to a conformational change in the protein at the binding site.