Conformations of nucleotides bound to wild type and Y78F mutant yeast guanylate kinase: proton two-dimensional transferred NOESY measurements.
Wild type and Y78F mutant yeast guanylate kinase (GKy) were studied to investigate the effects of a site-directed mutation on bound substrate conformations. Previously published work showed that Y78 is involved in GMP binding and that the Y78F mutant has 30-fold weaker GMP binding and 2 orders of magnitude less activity, than the wild type. Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) and guanosine conformations of guanosine 5'-monophosphate (GMP) bound to wild type and Y78F mutant yeast guanylate kinase in the complexes GKy x Mg(II)ATP, GKy x Mg(II)ADP, GKy x GMP, and GKy x Mg(II)ADP x [U-13C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations. For adenyl nucleotides in wild type complexes, all glycosidic torsion angles, chi, were 54 +/- 5 degrees. In Y78F mutant complexes, adenyl nucleotide glycosidic torsion angles were 55 +/- 5 degrees (GKy x MgATP) and 49 +/- 5 degrees (GKy x MgADP). Thus, the adenyl nucleotides bind similarly for both the wild type and Y78F mutant complexes. However, in the fully constrained, two-substrate complexes, GKy x Mg(II)ADP x [U-13C]GMP, the guanyl glycosidic torsion angle, chi, is 50 +/- 5 degrees with the wild type and 83 +/- 5 degrees with the Y78F mutant. This difference suggests that an unfavorable torsion may be a large part of the mechanism for significantly weaker GMP binding to reaction complexes of the Y78F mutant.
Ray
Bruce D
BD
Department of Physics, Indiana University-Purdue University at Indianapolis, 402 North Blackford Street, Indianapolis, Indiana 46202-3273, USA.
Jarori
Gotam K
GK
Raghunathan
Vidya
V
Yan
Honggao
H
Nageswara Rao
B D
BD
D000255:Adenosine Triphosphate / Q000378:metabolism; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D015003:Yeasts / Q000201:enzymology*
D009711:Nucleotides; D011522:Protons; D000255:Adenosine Triphosphate; D051528:Guanylate Kinases