Productive versus unproductive nucleotide binding in yeast guanylate kinase mutants: comparison of R41M with K14M by proton two dimensional transferred NOESY.

Because transfer of the terminal phosphoryl moiety from one mononucleotide to another, or from a mononucleotide either to a small molecule or to a protein substrate, plays a fundamental role in a number of aspects of metabolism, in signal transduction, and in gene regulation, much study has been invested in how kinases transfer phosphoryl groups (1). A synergistic interaction between site-directed mutagenesis, X-ray crystallography, and NMR spectroscopy has been found exceedingly helpful in elucidating structural concomitants of phosphoryl group transfer (2). An important component of the synergy has been determination of the degree of structural perturbation that results from a mutation, particularly in the substrate binding conformations. NMR has proven to be a useful experimental technique for solution phase determination of bound substrate conformations and distances of traversal of itinerant moieties (3-9). Productive conformations for a bi-substrate reaction are characterized by geometrical constraints such as the relative orientation of moieties within each bound substrate, the relative orientation of bound substrates with respect to each other, the orientation of each bound substrate with respect to the critical moieties of the enzyme, the traversal distances of itinerant substrate moieties, and other aspects regarding the topography of the active site. Guanylate kinase, by SCOP classification a member of the nucleotide and nucleoside kinases family in the P-loop containing nucleotide triphosphate hydrolases superfamily (10), is a small, typically monomeric enzyme that catalyzes the reversible reaction MgATP+GMP⇔MgADP+GDP (1) similar to the adenylate kinase reaction (11, 12). At 20.5 kD, yeast GK is part of a short sequence subfamily that lacks a full lid domain. Guanylate kinase's essential role in conversion of GMP to GDP, gives it a critical role in GTP biosynthesis. Consequently, cellular functions that involve GTP such as the control of the cGMP cycle (13) are affected by GK. GK's participation in the activation of antiviral acyclic guanosine analogs is a pharmacological consequence of its role in GTP biosynthesis (14-16), which biomedical role has attracted attention to the structure and function of guanylate kinase. Prior NMR studies of changes in bound substrate conformations between wild type and Y78F mutant GKy suggested significant perturbation of the glycosidic angle of bound GMP in the E•MgADP•GMP complex (3). Because of this significant perturbation on the substrate conformation, any straightforward mechanistic interpretation of kinetic and binding constant measurements of this mutant becomes less certain. Furthermore, this observation raises some additional questions. Because the hydroxyl of Y78 provides a hydrogen bond to the GMP phosphate, the perturbation observed might be a general effect of interference with hydrogen bonding to the phosphate. On the other hand, maintaining the bound GMP in a certain conformation might be a specific function of the hydroxyl of Y78. Finally, it might be an artifact of mutations within the GMP binding site not to be replicated in other nucleotide binding sites. Thus, the generality both of the observation, and of the method need further examination with some well selected GKy mutants. Furthermore, structural perturbation in this context should encompass the protein as well as the bound substrates. In yeast guanylate kinase, K14 is the “invariant lysine” of the canonical “P-loop” or Walker A (17) motif, GXXXXGKT/S, located in what is termed the core region of the enzyme (18-20). In contrast, R41 is part of the nucleotide monophosphate binding domain that contributes a hydrogen bond to the phosphoryl moiety of GMP (18, 20). Figure 1 shows the relative locations and orientations of K14 and R41 with respect to bound GMP as seen in X-ray crystallography (20). This paper presents a comparison of adenyl and guanyl conformations of enzyme-bound nucleotides with the K14M and R41M mutant enzyme of yeast guanylate kinase by means of TRNOESY. Figure 1 Relative locations of K14 and R41 with respect to GMP as revealed by X-ray crystallography (21). The R41 guanidino-GMP phosphate hydrogen bond is indicated by the dashed line. This figure was produced using the UCSF Chimera package from the Computer Graphics ...