Two-dimensional transferred nuclear Overhauser effect spectroscopy study of the confirmation of MgATP bound at the active and ancillary sites of rabbit muscle pyruvate kinase.
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 made at 500 MHz and 10 degrees C for several mixing times in the range 40-200 ms. The NOE values for the proton pair H1'-H2' of ribose (which are 2.9 +/- 0.2 A apart, irrespective of nucleotide conformation) as a function of ligand concentration (1-10 mM ATP), with the ratio of ligand to enzyme being kept constant, indicate that at higher ligand concentrations adventitious binding of ATP at nonspecific site(s) makes a major contribution to the observed NOEs. When the ligand concentration is < 2 mM, site-specific NOEs can be measured. Furthermore, addition of phosphoenolpyruvate (PEP) to the enzyme-MgATP sample results in competitive displacement of MgATP from the active site and reduces the observed NOE to that arising exclusively at the ancillary site, thus allowing the measurement of site-specific NOEs. The interproton distances determined from such site-specific NOE buildup curves were used as constraints in CHARMm to obtain the structure of MgATP. At the active site, MgATP has a glycosidic torsion chi = 44 +/- 5 degrees and the phase angle of pseudorotation for ribose P = 42.4 degrees. At the ancillary site chi = 46 +/- 5 degrees and P = 127.6 degrees. Thus the orientation of the adenine with respect to the sugar moiety is the same at both sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Jarori
G K
GK
Department of Physics, Indiana University-Purdue University at Indianapolis IUPUI 46202-3273.
Murali
N
N
Rao
B D
BD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008968:Molecular Conformation; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits
D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase