Structural Aspects of Manganese-Pyruvate Kinase Substrate and Inhibitor Complexes Deduced from Proton Magnetic Relaxation Rates of Pyruvate and a Phosphoenolpyruvate Analog

The distance between enzyme-bound Mn(II) at the active site of rabbit muscle pyruvate kinase and the protons of bound substrate or inhibitor was determined in ternary and quaternary enzyme complexes. In particular, the NMR relaxation rates of pyruvate protons effected by bound Mn(II) were measured for (a) the ternary pyruvate kinase-Mn(II)-pyruvate complex; (b) the abortive quaternary complex containing ADP as the fourth component; and (c) the quaternary complex containing ATP as the fourth component, i.e. the equilibrium mixture. The relaxation rates of the three proton resonances of the inhibitor, α-(dihydroxyphosphinylmethyl)acrylate, the methylene analog of P-enolpyruvate, were measured for the ternary enzyme-Mn(II)-inhibitor complex. Longitudinal (T1) and transverse (T2) NMR relaxation times were measured as a function of temperature and of Mn(II) concentration. Relaxation times were measured at two frequencies, 220 and 60 MHz, so that the correlation time and subsequently the distance of the substrate or inhibitor protons from the paramagnetic Mn(II) could be determined. The correlation time for all the pyruvate ternary and quaternary complexes was found to be 3.5 ± 1.2 x 10-9 s when determined from the T1:T2 ratio for the pyruvate resonance, and to be 2.7 x 10-9 s when determined from the frequency dependence of T1. The Mn(II) to pyruvate-proton distance of 8.2 ± 0.5 A is too large to accommodate a structure with direct Mn(II) coordination to pyruvate. The calculated distance is consistent with a structure in which the pyruvate is in the second coordination sphere of the Mn(II) bound to the enzyme. The correlation time for the ternary complex with the inhibitor, α-(dihydroxyphosphinylmethyl)acrylate, was found to be 1.0 x 10-9 s from the frequency dependence of T1. The distance from Mn(II), determined to be 5.3 A to the methylene protons, 6.5 A to one vinyl proton, and 6.2 A to the other vinyl proton, provides evidence that the ternary P-enolpyruvate analog complex involves coordination of the phosphinyl group to Mn(II) bound to the enzyme. Although binding experiments indicate that P-enolpyruvate and α-(dihydroxyphosphinylmethyl)acrylate compete for the same site on pyruvate kinase in the ternary complex, water proton relaxation rate data and EPR spectra show that the methylene analog of P-enolpyruvate forms a very different ternary complex than P-enolpyruvate. Consequently an extrapolation from the structure of the inhibitor complex to that of the substrate P-enolpyruvate is questionable.