Productive versus unproductive nucleotide binding in yeast guanylate kinase mutants: comparison of R41M with K14M by proton two dimensional transferred NOESY.
The R41M and K14M mutant enzymes of yeast guanylate kinase (GKy) were studied to investigate the effects of these site-directed mutations on bound-substrate conformations. Published X-ray crystal structures of yeast guanylate kinase indicate that K14 is part of the "P" loop involved in ATP and ADP binding, while R41 is suggested as a hydrogen bonding partner for the phosphoryl moiety of GMP. Both of these residues might be involved in transition state stabilization. Adenosine conformations of ATP and ADP and guanosine conformations of GMP bound to R41M and K14M mutant yeast guanylate kinase in the complexes GKy.MgATP, GKy.MgADP, and GKy.MgADP.[u-(13)C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations, and these conformations were compared with previously published conformations for the wild type. In the fully constrained, two substrate complexes, GKy.MgADP.[u-(13)C]GMP, the guanyl glycosidic torsion angle, chi, is 51 +/- 5 degrees for R41M and 47 +/- 5 degrees for K14M. Both are similar to the published 50 +/- 5 degrees published for wild type. For R41M with adenyl nucleotides, the glycosidic torsion angle, chi, was 55 +/- 5 degrees with MgATP, and 47 +/- 5 degrees with MgADP, which compares well to the 54 +/- 5 degrees published for wild type. However, for K14M with adenyl nucleotides, the glycosidic torsion angle was 30 +/- 5 degrees with MgATP and 28 +/- 5 degrees with MgADP. The results indicate that bound adenyl-nucleotides have significantly different conformations in the wild-type and K14M mutant enzymes, suggesting that K14 plays an important role in orienting the triphosphate of MgATP for catalysis.
Ray
Bruce D
BD
Department of Physics, Indiana University - Purdue University at Indianapolis (IUPUI), 402 North Blackford Street, Indianapolis, Indiana 46202-3273, USA.
Scott
Joshua
J
Yan
Honggao
H
Rao
B D Nageswara
BD
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D051528:Guanylate Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism
D011522:Protons; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D051528:Guanylate Kinases