H-1 AND P-31 RELAXATION RATE STUDIES OF THE INTERACTION OF PHOSPHOENOLPYRUVATE AND ITS ANALOGS WITH AVIAN PHOSPHOENOLPYRUVATE CARBOXYKINASE

The interactions of the substrate phosphoenolpyruvate and the substrate analogs (Z)- phosphoenol-.alpha.-ketobutyrate and (E)-phosphoenol-.alpha.-ketobutyrate with the enzyme-Mn complex of chicken liver phosphoenolpyruvate carboxykinase were investigated by 1H and by 31P nuclear relaxation rate studies. Studies of the 1H and the 31P relaxation rates of the ligands in the binary Mn-ligand complexes show that these ligands interact with the metal ion via the phosphate group but not through the carboxylate. An inner sphere coordination complex is formed but the metal-ligand complex is not in the most extended conformation. In the relaxation rate studies of the ligands in the presence of the enzyme, conditions were adjusted so that all of the Mn2+ that was added resided in the ternary enzyme-Mn-ligand complex. The 1H relaxation rates for each of the 3 ligands were measured at 100 and 300 MHz. In each case the normalized paramagnetic effects showed that 1/(pT2p) was greater than 1/(pT1p). A frequency dependence of the 1/(pT1p) and 1/(pT2p) values was also measured. The correlation time, .tau.c, for the Mn-1H interaction was calculated from the frequency dependence of 1/(pT1p) assuming a maximal frequency dependence of .tau.c and assuming no frequency dependence of .tau.c and from the T1M/T2M ratios at each frequency. The .tau.c values for all of the complexes, calculated at 100 MHz, varied from .apprx. 0.3-2.0 ns. These values were used to calculate the Mn-1H distances in each of the ternary complexes. The relaxation rates of 31P were also measured. The values of 1/(pT2p) were more than 1 order of magnitude larger than the respective values for 1/(pT1p) of 1H and of 31P for each ligand. A frequency dispersion, measured at 40.5 and at 121.5 MHz, was also observed for the 1(pT1p) values. A caculation of the Mn-P distances shows that phosphoenolpyruvate forms an outer sphere complex with the bound Mn2+ (r = 7.44 .+-. 0.52 .ANG.). The structures of the ternary complexes with the Z and E analogs (r = 3.74 .+-. 0.15 .ANG. and r = 4.79 .+-. 0.34 .ANG., respectively, where r is the Mn-P distance) demonstrate that these inhibitors bind differently to the enzyme than does the substrate. The exchange rate of phosphoenolypyruvate (koff = 2.2 .times. 104 s-1), measured from a temperature dependence of T2p, is several orders of magnitude greater than the turnover number (30 s-1) for the reaction. The activation energy for substrate exchange, 13 kcal/mol, suggests that this ligand exchange process is not a rate-determining step. These structural and kinetic results lead to a refinement of the proposed mechanism of this reaction and a clarification of the role of the Mn2+ activator.