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pH influences fluoride coordination number of the AlFx phosphoryl transfer transition state analog

Nature Structural Biology volume 6, pages 721–723 (1999)Cite this article

Phosphoryl transfer reactions are involved in processes such as energy transduction, signaling, regulation of cell growth and activation of metabolites. An important aspect of the action of these enzymes concerns the mechanism by which they transfer the phosphoryl group (mostly the γ-phosphate from ATP) to acceptors like water (hydrolases), amino acid residues (protein kinases) or other nucleotides (nucleoside monophosphate (NMP) kinases). The mechanism of phosphoryl transfer reactions can be studied using the transition state analog aluminum fluoride (AlFx), which has been opted for molecule of the year 1997 (ref. 1). A number of crystal structures of nucleotide binding proteins complexed with AlFx have been determined (Table 1). Surprisingly, many of these structures show AlF41-, although AlF3 mimics a phosphoryl group being transferred. Since a correlation of the crystallization conditions and the number of fluoride atoms can be observed (Table 1), we propose that the different coordination numbers of aluminum originate mainly from the difference in pH at which the enzymes were crystallized. To test this hypothesis, we determined the crystal structures of UMP/CMP-kinase from Dictyostelium discoideum (UmpKdicty) complexed with ADP, CMP and AlFx at pH 4.5 and pH 8.5, respectively. UmpKdicty is a nucleoside monophosphate kinase (ATP:NMP phosphotransferase) that catalyzes the reversible transphosphorylation between ATP and UMP or CMP (ref. 2) without forming a covalent enzyme intermediate3.

Table 1 Comparison of coordination numbers at different crystallization conditions
Full size table

Structures of transition state analog complexes of phosphoryl-transferring enzymes can be used to deduce whether the mechanism of the phosphoryl transfer reaction is associative or dissociative4. Since the two mechanisms differ in charge distribution and bond order of the transition state, the location of positively charged amino acids and the distance of the phosphoryl analog to the leaving and attacking groups are crucial. In the case of UmpKdicty the mechanism was deduced to be associative on the basis of the adjacency of five arginine residues to the AlF3 group5. Interestingly, the distances between AlF3 and the phosphate groups of ADP and CMP, respectively, are greater than the corresponding ones in the AlF41- complex (Fig. 1). This finding indicates that the precise geometry of the active sites of phosphoryl-transferring enzymes may depend on factors that are not necessarily connected to the mechanism itself. Although it was possible to deduce correctly the general principle of the mechanism, specific interactions may change when switching from AlF41- to AlF3. In the case of UmpKdicty, the most pronounced change of the active site geometry concerns Arg 93, which interacts more strongly with one of the fluorides of AlF3 than with AlF41- (Fig. 1). Mutagenesis studies indicate that this interaction is important, since substitution of this arginine results in a 100- to 1,000-fold reduced catalytic rate6,7.

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Figure 1: Electron density maps and AIFx models.

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Acknowledgements

We thank R.S. Goody and R.M. Sweet for continuous and encouraging support. The facility used for data collection is supported by the US Department of Energy, Offices of Health and Environmental Research and of Basic Energy Sciences, and by the National Science Foundation. We thank J. Cherfils for communicating the second possible explanation for the pH switch in AlFx coordination. J.S. is grateful for the generous support by the Richard und Anne-Liese Gielen-Leyendecker-stiftung.

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  1. Max Planck Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, Postfach 500247, D-44227, Dortmund, Germany

    Ilme Schlichting & Jochen Reinstein

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Correspondence to Ilme Schlichting or Jochen Reinstein.

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Schlichting, I., Reinstein, J. pH influences fluoride coordination number of the AlFx phosphoryl transfer transition state analog. Nat Struct Mol Biol 6, 721–723 (1999). https://doi.org/10.1038/11485

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