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Title: Kinases : charge balance and structural tightening in the transition state
Author: Marston, James Paul
ISNI:       0000 0004 2720 5663
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2010
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Phosphoryl transfer reactions are ubiquitous in biology, and metal fluoride TSA complexes have played a key role in structural approaches to understanding how they are catalysed. These complexes have permitted experimental tests of two hypotheses: the proposal that the balance of charge in the transition state dominates in enzyme catalysed phosphoryl transfer; and the proposal that structural tightening contributes to transition state stabilisation. In this thesis, the nature of the catalytically relevant state of PGK was revealed from analysis of new 19F NMR data, and the previously determined high resolution X-ray structures of the MgF3- and AIF4--TSA complexes. The sidechain of K219, which coordinates the a-phosphate in ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphate. Consistent with the dominance of charge balance in transition state organisation, the substitution K219A induces a corresponding reduction in the charge of the metal fluoride species of the TSA, forming an AIF 3 ° complex. The octahedral geometry of the AIF30 species points to widespread misassignments of MgF3- complexes as AIF30 complexes. An AIF4--TSA complex of the p38a MAPK:MKK6 heterodimer was also detected by 19F NMR. The metal fluoride moiety is isoelectronic with the migrating phosphoryl group: a requirement hitherto only demonstrated for proteins having a single catalytic Mg2+. Changes in backbone amide chemical shifts and HID exchange protection were measured for the substrate, product and TSA bound states of PGK to assess whether they are consistent with changes in hydrogen bonding within the protein. A global structural tightening mechanism was not supported by the data, as no overall shortening of hydrogen bonds could be demonstrated. Instead, data consistent with structural tightening were confined to the PGA binding site and hinge regions, reinforcing existing models of cooperativity and interdomain communication in PGK.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available