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Access to Electronic ThesisAuthor:Hugh DannattThesis title:The Role of Enzyme Dynamics in Catalysis by ß-PhosphoglucomutaseQualification:PhDThis electronic thesis is protected by the Copyright, Designs and Patents Act 1988.No reproduction is permitted without consent of the author. It is also protected bythe Creative Commons Licence allowing Attributions-Non-commercial-Noderivatives.This thesis was embargoed until 13th September 2015.If this electronic thesis has been edited by the author it will be indicated as such on thetitle page and in the text.

The Role of Enzyme Dynamics inCatalysis by β-PhosphoglucomutaseHugh R. W. DannattDepartment of Molecular Biology & BiotechnologyThe University of SheffieldThesis submitted for the degreeof Doctor of PhilosophyAugust 2012

AbstractThis thesis primarily concerns the use of nuclear magnetic resonance (NMR) spectroscopy to study enzyme dynamics. Recent improvements in NMR and other biophysical methods have allowed the detailed study of protein dynamics, and have ledto much speculation as to their involvement in the catalytic prowess of enzymes.β-Phosphoglucomutase (βPGM) is a phosphoryl transfer enzyme, and must therefore bring one of the slowest chemical reactions in nature onto a biologically-relevanttimescale. As well as being exceptionally proficient catalysts, phosphate transfer enzymes are useful objects of study through the use of metal fluorides and ground-stateand transition-state analogues which allow for the capture of conformations which relate to various stages along the reaction coordinate. Together then, βPGM and metalfluorides allow for a critical analysis of the role of enzyme dynamics in catalysis.Formation of the transition state analogue (TSA) complex of βPGM captured byusing MgF3 ions to mimic the transferring phosphate allows the study of both protein& TSA NMR signals. It is demonstrated that there are two conformational exchangeprocesses occurring on the catalytic timescale: one which is coincident in rate to catalysis and therefore likely plays a role in catalytic turnover by βPGM, and another whichis implicated in the folding stability of the protein. These processes are characterisedin detail, and attempts to perturb them by mutation are made. The role of dynamicsoccurring on a faster timescale in βPGM is also explored.It is concluded that in βPGM and all other enzymes, dynamics on the timescalesmost amenable to study by NMR (ms–µs and ns–ps timescales) are not directly involvedin the chemical step. Instead they may be involved in allowing the enzyme to traversethe complex energy landscape from substrate to product, a role of equal importance.i

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AcknowledgementsI must first thank my supervisor Jon Waltho for correctly assessing during myinterview that I was game for a challenge and choosing my PhD project accordingly.Despite my luck rarely matching my imagination, Jon has always allowed me to exploremy ideas and spend the necessary time getting to grips with theory and experimentalprocedure so as to achieve the best results possible. There were plenty of times —whilst undertaking certain unspecified duties — that he would welcome any legitimateprocrastination and was always able to provide insight no matter how cloudy the view.I have also been allowed to attend a near-excessive number of conferences during mystudies, which has exposed me to many new concepts and people, as well as plentifulfree alcohol. For all this I am very grateful.Matt has been absolutely indispensable during my time in the NMR group. It israre to find someone to be so experienced and carefully considered in both practicalprocedures and data handling that you can have 100% confidence that whatever theyhave told you is right. It is also rare to find someone equally cynical and committedto doing things “the right way” as I have tried to be throughout. As a result Matt hasbeen my sounding board throughout my PhD. I have come to trust and rely on hisadvice on all matters scientific, and even for things concerning life outside of the lab.I will try hard to resist the urge to continue sending frequent e-mails once I have leftthe group, but his validation will be missed!I never ceased to be amazed by the seemingly endless catalogue of βPGM experiments & data that Nicky could pull out of her many lab books, making her animmensely valuable consultant for all things βPGM. Her keen eye for detail and abilityto write the most fantastically transparent and unambiguous e-mails even when describing extremely complex data are amongst several traits that I hope have rubbed offon me. Despite being a physicist by training, Jeremy was always able to really graspwhere the difficulties lie in understanding NMR and other more mathematical problems from the point-of-view of a biochemist, which makes him a talented and effectiveteacher. His scepticism and ability to see through jargon and ill-conceived ideas werealso extremely useful providing they weren’t exercised whilst I was giving a talk!Mike has an unparallelled knowledge of all things protein-related and would alwayshumour me when I asked about NMR techniques that I will likely never perform butwould nonetheless like to understand. The same goes for Andrea who would alwaysanswer questions about how the magnets are built, how much they cost to run, whathappens during a quench, etc, etc.! Her help for setting up complicated pulse programs,particularly at the start of my PhD, was invaluable. I particularly appreciated Maya’sguidance on setting up and processing the data from the relaxation experiments, I’mglad I was able to take your dispersion pulse program on tour! I should also thank Lazfor managing to install Relax on one of our machines remotely, even though I couldn’tdo it locally!iii

As fellow βPGM sufferers (although in actual fact it is a wonderful little protein),Jo & Jin Yi have made excellent company, always keen not to take things too seriouslyand provide guidance in the lab. Recovering from injury and having not been near awet lab for the best part of 18 months, to describe me as rusty for the first proteinprep would be a huge understatement! I wish the best of luck to Luke, with whom itseems the project is in safe hands. With decreasing numbers of people around, I havecome to rely more and more on Pete to cheer me up and engage in a bit of (mostly)harmless mischief. His donation to the wet lab of some computer speakers has beeninstrumental in allowing me to retain some sanity during dreaded buffer exchanges,albeit at the expense of everyone else’s. The latest batch of ice cream was particularlygood, and “Falafel Tuesdays” is a recent tradition that I will definitely miss. You canhave my Yttrium Chloride, Pete, use it wisely! (Don’t use it at all!)Shrimp deserves a mention even if just for the note she once left on my bench: “theindian people got your sample”, which will never not be absolutely hilarious. I alsothank Yasuko for preventing the complete destruction of my lab bench at the hands ofa rogue bunsen burner. Ultimately how much I have enjoyed the time in the lab hasbeen largely down to the people in it, and so in no particular order I would also liketo thank: Abi, Rob, Caz, Clare, Dave, Andy, Amie, Jenny, James & Farhat for theircompany. At risk of going off on a tangent, I am also indebted to the NMR DiscussionGroup, being invited to give 2 talks and having them so well received was an enormousconfidence boost for me last year.Not wanting to waste more tree than is necessary (good job I’m printing doublesided as this already resembles an oscar acceptance speech), I would like to brieflyacknowledge a few key groups of people outside of the lab who have made the last 4years what they have been. Firstly, the housemates I have been lucky to live with whilsthere in Sheffield, particularly Cooney & John who have lived in Sheffield throughout.Secondly, anyone with whom I’ve been lucky enough to play music, but of course specialmention goes to Sam B., Sam K., Tom & Scott. Thirdly, the members of the PostgradSociety & PG Café Forum, and especially the fantastic people who helped run thecommittees whilst I sat on them and those who have continued to do so since. Asfor everyone else, in the exceedingly unlikely event that you read this: you know whoyou are and I expect assistance with the further punishing of my liver when the timecomes.My parents have been the best support anybody could hope for. Not once did thewords “why don’t you get a real job?” leave their mouths, and I dare say it neverentered their heads. It is easy to get caught up in the technicalities and details andwhat’s going on around you, but they were always on hand to remind me to step backand get enthused about the things that mattered, often getting more excited than I wasabout whatever news I had to share. My Dad’s relentless attempts to understand whaton earth I was talking about were heroic (entirely my fault, not his!). The supportthat I have received from my parents over the last few months in particular has beeniv

immense — well above and beyond the call of duty — and I am truly grateful. I alsothank Soph & Mark for their support, and in particular putting me up so many timesand often taking me to the airport to make travelling around to conferences or otherEuropean labs so much easier. I feel right at home in your spare room, and I hope thiswill continue!Last, and certainly not least, Becks. I cannot begin to thank her for everythingshe has done for me, I don’t want to write another thesis. Her unyielding faith in meborders on delusional I’m sure. Through the more difficult times during my PhD I havebeen awarded more patience than anyone deserves, and she always had the energy togive me a pep talk, even if I didn’t have the energy to listen. If she was less generousand kind than she is, just seeing her face on a daily basis would have been supportenough. Thank you.Right. Apart from the bloody abbreviations list, I’m done. Phew.Hugh Dannatt10th August 2012, 2:51 amv

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