Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes 1H-transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase.
The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 1H NMR. The experiments were performed on D2O solutions at 276 MHz and 300 MHz, and 10 degrees C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase-alternating hard 1.8 degree pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6-8 delay times ranging from 10-500 ms following the DANTE sequence. A full complement of ten NOE build-up curves obtained for each enzyme complex was analyzed by using the complete relaxation-matrix method (which includes all the non-exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE-determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3'-endo sugar pucker and an anti conformation with a glycosidic torsional angle (O'4-C'1-N9-C8) of 39 degrees +/- 4 degrees. Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth-power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P.R., Fox, T.L. & Mildvan, A.S. (1987) Biochemistry 26, 3487-3493.
Landy
S B
SB
Department of Physics, Indiana University-Purdue University, (IUPUI), Indianapolis 46205-2810.
Ray
B D
BD
Plateau
P
P
Lipkowitz
K B
KB
Rao
B D
BD
D000225:Adenine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D008718:Methionine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D008968:Molecular Conformation; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism
D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008718:Methionine-tRNA Ligase; D000225:Adenine