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.Mg(II)ATP, GKy.Mg(II)ADP, GKy.GMP, and GKy.Mg(II)ADP.[U- 1 3 C]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- 1 3 C]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.