Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.678092
Title: Structural and mechanistic investigation of enzyme-catalysed phosphoryl transfer in two HAD superfamily proteins
Author: Johnson, Luke A.
ISNI:       0000 0004 5370 0297
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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Abstract:
Metal fluoride analogues provide isosteric and isoelectronic mimics for the transition and ground states of phosphoryl transfer. In combination with 19F NMR, these serve as direct probes for the electronic environment of the catalytic site. The work presented within this thesis focuses on using metal fluoride analogues, together with site directed mutagenesis and biological NMR, to further understand how phosphoryl transfer is performed by two HAD superfamily enzymes; β-phosphoglucomutase and phosphoserine phosphatase. Using fluoroberyllate analogues, the ground states of βPGM were investigated, establishing that at high pH, BeF2OH- replaces BeF3- as a phosphoryl transfer analogue. Importantly, the BeF2OH- moiety preferentially binds βG1P, allowing for study of the ground state for step 1 of βPGM's reaction coordinate, previously inaccessible. This further established the molecular details for how βPGM performs catalysis on both orientations of the hexose, and highlights how hydrogen bonds between the substrate and the phosphate enables the nucleophile to approach the anionic phosphate in the absence of the general base. Separately, an intermediate complex of βPGM was established through mutation of the general base. Kinetic investigation reveals that the D10N mutant is catalytically active, yet after exposure to substrates a βG1,6bisP complex forms. Taken with fluoroberyllate and fluoromagnesate analogue complexes of βPGM, this demonstrates once again how phosphoryl transfer involves in-line nucleophilic attack, with an associative concerted mechanism, where the phosphorous atom migrates 1.2 Å between axial oxygen atoms. A full backbone assignment was performed, and ms - µs dynamics investigated by 15N relaxation dispersion. Lastly, the functional importance of structural differences between βPGM and PSP were explored by site directed mutagenesis. Substrate and product complexes were formed with D13N PSP, confirming the generally applicability of this general base mutation for studying trapped states in enzyme-catalysed phosphoryl transfer. Meanwhile, assignment of the MgF3- TSA complex for the hydrolysis reaction was completed, and dynamics of PSP for apo and MgF3- TSA species probed.
Supervisor: Waltho, Jonathan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.678092  DOI: Not available
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