Productive versus Unproductive Nucleotide Binding in Yeast Guanylate Kinase Mutants: Comparison of R41M with K14M by Proton Two Dimensional Transferred NOESY

• Published in: Biochemistry (Easton) 2009-06, Vol.48 (24), p.5532-5540
• Main Authors: Ray, Bruce D, Scott, Joshua, Yan, Honggao, Rao, B. D. Nageswara
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Binding Sites; Guanylate Kinases - chemistry; Guanylate Kinases - genetics; Guanylate Kinases - metabolism; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Mutation; Protons; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi900139a

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-13C]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-13C]GMP, the guanyl glycosidic torsion angle, χ, is 51 ± 5° for R41M and 47 ± 5° for K14M. Both are similar to the published 50 ± 5° published for wild type. For R41M with adenyl nucleotides, the glycosidic torsion angle, χ, was 55 ± 5° with MgATP, and 47 ± 5° with MgADP, which compares well to the 54 ± 5° published for wild type. However, for K14M with adenyl nucleotides, the glycosidic torsion angle was 30 ± 5° with MgATP and 28 ± 5° 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.