CONFORMATION OF MGATP BOUND TO 5-PHOSPHO-ALPHA-D-RIBOSE 1-DIPHOSPHATESYNTHETASE BY 2-DIMENSIONAL TRANSFERRED NUCLEAR OVERHAUSER EFFECT SPECTROSCOPY

The conformation of MgATP bound at the active site of Salmonella typhi murium 5-phospho-alpha-D-ribose 1-diphosphate synthetase (PRibPP synth etase) has been investigated by two-dimensional transferred-NOE spectr oscopy (TRNOESY). Inter-proton NOEs of the Ligand were measured in the presence of the protein at several mixing times in the range of 40-30 0 ms at 500 MHz and 10 degrees C. Measurements were made at low ligand concentrations (approximate to 1 mM) in order to avoid weak non-speci fic Ligand-protein interactions and to ensure that the NOE arises from the ligand bound at the active site. The inter-proton distances were determined from the experimentally observed NOE buildup curves by comp aring them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecu lar modeling and energy minimization calculations to deduce the struct ure of the bound ligand. PRibPP synthetase is known to appreciably agg regate so that it exists in multiple oligomeric forms in solution. The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size. On the basis of the rotational correlation time deduced for the enzyme-nucleotide complexes, it is estimated that the average oli gomer of PRibPP synthetase, in the sample used for the TRNOESY measure ments, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer. The conformation of enzyme-bound MgATP is described by a glycosidic torsion angle chi = 50+/-5 degrees and ph ase angle of pseudorotation P = 114.9 degrees corresponding to a T-1 d egrees sugar pucker. It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of cre atine kinase, pyruvate kinase (active and ancillary sites), and argini ne kinase. The sugar pucker, on the other hand, differs from one enzym e complex to another.