Transition state analogue structures of human phosphoglycerate kinase establish the importance of charge balance in catalysis.
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 (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-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 alpha-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 (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).
Cliff
Matthew J
MJ
The Krebs Institute & The Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Bowler
Matthew W
MW
Varga
Andrea
A
Marston
James P
JP
Szabó
Judit
J
Hounslow
Andrea M
AM
Baxter
Nicola J
NJ
Blackburn
G Michael
GM
Vas
Mária
M
Waltho
Jonathan P
JP
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D017607:Aluminum Compounds / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D055592:Biophysical Phenomena; D004583:Electrons; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism; D005988:Glyceric Acids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary
D017607:Aluminum Compounds; D005988:Glyceric Acids; D007527:Isoenzymes; C050992:tetrafluoroaluminate; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase; D008274:Magnesium; D005459:Fluorides