Enzyme Catalysed Phosphoryl Transfer Reactions: Magnesium and Other Metal Fluorides as Substrate and Transition-State Analogs

George M. Blackburn 

Sheffield University, Chemistry Department, Brook Hill, Sheffield S37HF, United Kingdom

Abstract

Magnesium trifluoride, MgF3-, has been assigned [1, 2] and under-assigned [3] as the molecular species seen in x-ray structures of trigonal-bipyramidal species (tbp) of enzymes catalysing phosphoryl transfer. A structure of ß-phosphoglucomutase (ßPGM) complexed with G6P has been presented as a pentacovalent tbp phosphorus intermediate [3]. Our alternative attribution of MgF3- as the tbp species [4] has been strongly rejected [5].

Kinetic, spectroscopic, and structural data confirms MgF3- as a TSA for ßPGM. F- is a strong inhibitor for ßPGM and 19F NMR analysis shows three F- ions are recruited into the active site of the enzyme in the presence of G6P. NOE analysis using 15N,2H-labelled ßPGM shows 4 amino acids in close contact with two F-; the third F- is co-ordinated to a catalytic Mg2+ ion. This accurately locates the binding site for MgF3- which is further co-ordinated to G6P C1-OH group and to Asp8. In examples of metal fluoride complexes of phosphoryl transfer enzymes identified by x-ray analysis, the presence of F- (rather than OH-) must be inferential at the resolutions achieved. Our results constitute the first direct observation of a metal fluoride complex as a TSA for a phosphoryl transfer enzyme. The bonding in the tbp accurately portrays the transition state for phosphoryl transfer in ßPGM and reveals the details of protein catalysis.

[1] D. L. Graham, P. N. Lowe et al., Chem. Biol. 2002, 9, 375-381.
[2] M. F. Bowler, G. M. Blackburn, A. G. W. Leslie, J. E. Walker, Abstr. Gordon Conf. Biophys. 2005.
[3] S. D. Lahiri, G. Zhang, D. Dunaway-Mariano et al., Science (Washington, DC, USA) 2003, 299, 2067-2071.
[4] G. M. Blackburn, N. H. Williams et. al., Science (Washington, DC, USA) 2003, 301, 1184.
[5] L. W. Tremblay, G. Zhang et al., J. Am. Chem. Soc. 2005, 127, 5298-5299.

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Presentation: oral at 18th Conference on Physical Organic Chemistry, Symposium 1, by George M. Blackburn
See On-line Journal of 18th Conference on Physical Organic Chemistry

Submitted: 2006-05-19 14:06
Revised:   2009-06-07 00:44
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