Conformation of MgATP bound to 5-phospho-alpha-D-ribose 1-diphosphate synthetase by two-dimensional transferred nuclear Overhauser effect spectroscopy.
The conformation of MgATP bound at the active site of Salmonella typhimurium 5-phospho-alpha-D-ribose 1-diphosphate synthetase (PRibPP synthetase) has been investigated by two-dimensional transferred-NOE spectroscopy (TRNOESY). Inter-proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40-300 ms at 500 MHz and 10 degrees C. Measurements were made at low ligand concentrations (approximately 1 mM) in order to avoid weak non-specific 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 comparing them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand. PRibPP synthetase is known to appreciably aggregate 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 oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, 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 phase angle of pseudorotation P = 114.9 degrees corresponding to a 1T degree 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 creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase. The sugar pucker, on the other hand, differs from one enzyme complex to another.
Jarori
G K
GK
Department of Physics, Indiana University Purdue University Indianapolis 46202-3273, USA.
Murali
N
N
Switzer
R L
RL
Rao
B D
BD
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology
D000255:Adenosine Triphosphate; D012268:Ribose-Phosphate Pyrophosphokinase