Two-Dimensional Transferred Nuclear Overhauser Effect Spectroscopy Study of the Conformation of MgATP Bound at the Active and Ancillary Sites of Rabbit Muscle Pyruvate Kinase

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Abstract
Pyruvate,kinase,binds,one,adenosine,5’-triphosphate (ATP) molecule,at its active site and another,at an ancillary,site on each,subunit. In order to determine,the conformation,of ATP bound,at these sites, proton transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements were($#$R., Fox, T. L., & Mildvan, A. S. (1987) Biochemistry 26, 3487-3493; Landy, S. B., Ray, B. D., Plateau, P., Lipkowitz, K. B., & Nageswara Rao, B. D. (1 992) Eur. J. Biochem. 205,59-69). In both these studies the experiments were performed at high ligand concentrations, at which the observed NOEs are dominated by MgATP bound,at weak,nonspecific,binding,sites. Thus specific displacement,of MgATP from,the active site by the addition,of PEP did not significantly,alter the observed,hOEs. By the same,reasoning,the NOE data in these studies could not provide correct conformational information. In the experiments presented here, the contribution,from,adventitious,ligand binding,at the nonspecific,site(s) was,circumvented,by selecting appropriate,ligand,and,protein concentrations.,This experimental,protocol,has led to the determination,of the site-specific conformations,of MgATP at the active and,ancillary,sites on the enzyme. Pyruvate kinase is one of the two,enzymes,that play the

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