Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260724
Title: Cryocrystallographic and mechanistic studies on glycogen phosphorylase
Author: Mitchell, Edward Peter
ISNI:       0000 0001 3411 6210
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1994
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Abstract:
Glycogen phosphorylase (GPb) regulates the degradation of glycogen to glucose-1-phosphate and catalyses the first step of the reaction. Many studies have provided insights into the essentials of the catalytic mechanism. Previous time resolved crystallographic work using heptenitol has revealed a putative phosphate binding site at the active site of phosphorylase. Using nojirimycin tetrazole, a transition state analogue, complexed with phosphate and both T and R state GPb crystals, this work has conclusively located the phosphate binding site and the concomitant active site conformational changes. This has confirmed the previous heptenitol results. Using R state crystals complexed with tetrazole and phosphate, data were collected to 2.5Å resolution, higher than for the original 2.8Å resolution native structure, and used to position water molecules in the R state model. Further to this, direct observation of the phosphate ion orientation was made possible using flash-frozen T state crystals to collect 1.7Å resolution data at 100K. As part of this cryocrystallographic work the relationship of cryoprotectant concentration with crystal mosaicity was established, aiding the systematic search for flash-freezing cryoprotectant conditions for all protein crystals. Collection of a new native T state data set to 1.5Å resolution was made possible using the flash-freezing technique. Refinement has produced a new higher precision native model (R factor currently 22.8%) containing additional N terminal residues (14-18) and 330 new water molecules. A molecule of glycerol, the cryoprotectant, was located at the active site. This study represents a considerable improvement over the 1.9Å resolution room temperature native data, and is also the first time such high resolution data have been collected from such a large enzyme. In a further analysis of the phosphate binding properties, a link between structure, atomic charges and the ability of a ligand to bind phosphate at the phosphorylase active site was established. In order of phosphate binding ability, the nojirimycin tetrazole, nitroglucal, glucal and glucose complexes with T state GPb and phosphate were structurally analysed. As the charge difference between the pyranose ring oxygen (or equivalent atom) and anomeric atom becomes more negative (charges estimated using MOPAC) the tendency to bind phosphate decreases. The ligand must possess a half-chair conformation as this is essential to bind phosphate; glucose, having the most negative charge difference and a full chair conformation, does not bind phosphate significantly in the crystal. A novel surface binding site for nitroglucal (covalently linked to His 73) was also located during this work. As part of an ongoing search for potential drugs for diabetes, two gluco-hydantoin inhibitors of GPb were investigated. One proved to be the best inhibitor to date, and the inhibition was rationalised using the structural results from this work. A new improved inhibitor has been proposed on the basis of these results.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.260724  DOI: Not available
Keywords: Enzyme crystallography
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