Transition State Analogue Structures of Human Phosphoglycerate Kinase Establish the Importance of Charge Balance in Catalysis

• Published in: Journal of the American Chemical Society 2010-05, Vol.132 (18), p.6507-6516
• Main Authors: Cliff, Matthew J, Bowler, Matthew W, Varga, Andrea, Marston, James P, Szabó, Judit, Hounslow, Andrea M, Baxter, Nicola J, Blackburn, G. Michael, Vas, Mária, Waltho, Jonathan P
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Aluminum Compounds - chemistry; Aluminum Compounds - metabolism; Biocatalysis; Biophysical Phenomena; Electrons; Fluorides - chemistry; Fluorides - metabolism; Glyceric Acids - chemistry; Glyceric Acids - metabolism; Humans; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Magnesium - chemistry; Magnesium - metabolism; Models, Molecular; Phosphoglycerate Kinase - chemistry; Phosphoglycerate Kinase - genetics; Phosphoglycerate Kinase - metabolism; Point Mutation; Protein Structure, Tertiary
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja100974t

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF3 −) and tetrafluoroaluminate (AlF4 −) complexes of PGK have been determined using X-ray crystallography and 19F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the α-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF3 0) complex. The AlF3 0 moiety retains the octahedral geometry observed within AlF4 − TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF3 0 complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF3 −.