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. 2009 Jun 23;48(24):5532-40.
doi: 10.1021/bi900139a.

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

Bruce D Ray  1 , Joshua Scott, Honggao Yan, B D Nageswara Rao
Affiliations

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

Bruce D Ray et al. Biochemistry. .

Abstract

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.

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Figures

Figure 1
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 Laboratory, University of California, San Francisco, CA (supported by NIH Grant P41 RR-01081) (22).
Figure 2
Figure 2
Chemical structure of adenosine moiety showing the numbering for the relevant protons and the torsion angles. Torsions are denoted as follows: χ (O4′-C1′-N9-C8), ν0 (C4′-O4′-C1′-C2′), ν1 (O4′-C1′-C2′-C3′), ν2 (C1′-C2′-C3′-C4′), ν3 (C2′-C3′-C4′-O4′), ν4 (C3′-C4′-O4′-C1′), γ (O5′-C5′-C4′-C3′).
Figure 3
Figure 3
Comparison of conformations of ADP in the E•MgADP complex for wild type (red) and K14M mutant (blue) GKy. Each of the structures drawn represents one of the acceptable structures obtained for the respective complex by energy minimization.
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