ADENOSINE CONFORMATIONS OF NUCLEOTIDES BOUND TO METHIONYL TRANSFER-RNA SYNTHETASE BY TRANSFERRED NUCLEAR OVERHAUSER EFFECT SPECTROSCOPY

The conformations of MgATP and AMP bound to a monomeric tryptic fragme nt of methionyl tRNA synthetase have been investigated by two-dimensio nal proton transferred nuclear Overhauser effect spectroscopy (TRNOESY ). The sample protocol was chosen to minimize contributions from adven titious binding of the nucleotides to the observed NOE. The experiment s were performed at 500 MHz on three different complexes, E.MgATP, E.M gATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distance s obtained by fitting NOE build-up curves (not involving H5' and H5'') were used to determine a CHARMm energy-minimized structure. The posit ioning of the H5' and H5'' protons was determined on the basis of a co nformational search of the torsion angle to obtain the best fit with t he observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curv es for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three compl exes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a T-3(4)-E-4 sugar pucker, The glycosidic torsion angle, X, deduced here for this adenylyl transfer enzyme and those determined previously for three pho sphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruva te kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values sug gests a possible common motif for the recognition and binding of the a denosine moiety at the active sites of ATP-utilizing enzymes, irrespec tive of the point of cleavage on the phosphate chain.