Nuclear Overhauser effect studies of the conformations of magnesium ATP bound to the active and secondary sites of muscle pyruvate kinase

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Abstract
MgATP binds both at the active site (site 1) and at secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (.chi.=68 .+-. 10.degree.) and a C3''-endo ribose pucker (.delta.=90 .+-. 10.degree.). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 .+-. 10% low anti (.chi. = 30.degree.), C3''-endo; 20 .+-. 8% high anti (.chi.=55.degree.), O1''-endo; and 18 .+-. 8% syn (.chi.=217.degree.), C2''-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., and Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O''Brian, C., Kaiser, E. T., and Mildvan, A. S. (1983) Biochemistry 22, 3439]. The unusual multiplicity of adenine-ribose conformations of ATP bound at the active site of pyruvate kinase is consistent with the low specificity of this enzyme for nucleotide substrates [Plowman, K. M., and Krall, A. R. (1965) Biochemistry 4, 2809] and with the high mobility of bound 1,N6-ethenoadenosine 5''-triphosphate [Barrio, J. R., Secrist, J. A., Chien, Y. Taylor, P. J., Robinson, J. L., and Leonard, N. J. (1973) FEBS Lett. 29, 215].