Conformations of Nucleotides Bound to Wild Type and Y78F Mutant Yeast Guanylate Kinase:  Proton Two-Dimensional Transferred NOESY Measurements

• Published in: Biochemistry (Easton) 2005-10, Vol.44 (42), p.13762-13770
• Main Authors: Ray, Bruce D, Jarori, Gotam K, Raghunathan, Vidya, Yan, Honggao, Nageswara Rao, B. D
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Guanylate Kinases - genetics; Guanylate Kinases - metabolism; Models, Molecular; Mutagenesis; Nuclear Magnetic Resonance, Biomolecular - methods; Nucleic Acid Conformation; Nucleotides - chemistry; Nucleotides - metabolism; Protons; Yeasts - enzymology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0509088

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·Mg(II)ATP, GKy·Mg(II)ADP, GKy·GMP, and GKy·Mg(II)ADP·[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, χ, were 54 ± 5°. In Y78F mutant complexes, adenyl nucleotide glycosidic torsion angles were 55 ± 5° (GKy·MgATP) and 49 ± 5° (GKy·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·Mg(II)ADP·[U-13C]GMP, the guanyl glycosidic torsion angle, χ, is 50 ± 5° with the wild type and 83 ± 5° 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.