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Mechanistic and structural study of GAP-catalysed hydrolase activity in small G proteins

Baumann, Patrick 2021. Mechanistic and structural study of GAP-catalysed hydrolase activity in small G proteins. PhD Thesis, Cardiff University.
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Abstract

Small G proteins are a multifacetted class of enzymes and a major hub for signal transduction. In humans, dysregulation of this process can be the driver of a myriad of diseases, most prominently cancer. The first part of this work examines the newly discovered cSrc-mediated Switch-phosphorylation of the small G protein Ras. Conflicting literature reports exist regarding the Ras phosphorylation site and the impact the phosphorylation has on the interaction with its effectors. This work identifies Tyr64 to be the primary target of cSrc and examines structural implications the Ras phosphorylation has on the interaction with its effector RasGAP. Furthermore, this chapter provides an updated model for the only available HRas/RasGAP crystal structure and adresses misconceptions about this structural model, which is still widely used for computational studies. In the second part of this project the hydrogen bonding network of the small G protein RhoA in the transition state analog complex with its RhoGAPR85A effector is investigated. Through the application of a polyspecific tRNA/tRNA synthetase pair, a series of site-specifically labelled fluorotyrosine-RhoA variants were generated. This enables the selective tuning of a single hydrogen bond, donated by Tyr34 to the transfering phosphoryl group. This effect was quantified by investigating the MgF3 – transition state analogue complex between the fluorotyrosine-RhoA variants and RhoGAPR85A using 19F-NMR. Protein X-ray crystallography confirmed that the active site integrity was unperturbed by the fluorotyrosine incorporation. The kinetic parameters for the GTP hydrolysis of the fluorotyrosine-RhoA variants were measured using an HPLC-based assay under single-turnover conditions. Together these data reveal an impaired ability to stabilise the build-up of negative charge during the phosphoryl transfer transition state with decreasing Tyr34 hydroxyl pKa. This has implications for related systems such as the small GTPase Ran and establishes a methodology for probing single hydrogen bonds in enzyme active sites

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 27 May 2022
Last Modified: 27 May 2022 13:24
URI: https://orca.cardiff.ac.uk/id/eprint/150076

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