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Structural Studies of Catalysis and Processing in Galactose

Akumanyi, Nana (2007) Structural Studies of Catalysis and Processing in Galactose. PhD thesis, University of Leeds.

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Abstract

The fungal copper containing enzyme galactose oxidase is one of the earliest examples of a protein derived radical cofactor. A covalent bond, formed between the active site cysteine and the Cc of a tyrosine act as a site for radical formation, allowing the enzyme to catalyse the two electron oxidation of primary alcohols to their corresponding aldehydes, with the coproduction of hydrogen peroxide. The formation of the post-translationally modified redox cofactor is an autocatalytic process in vitro, requiring only the aerobic addition of copper. A premature form of galactose oxidase, lacking the cofactor has been crystallised and the structure determined. Copper incubation of this protein has been carried out and the structures determined in order to characterise processing intermediates and the mechanism of cofactor biogenesis. The mature, fully processed and catalytically active enzyme contains a radical which is thought to be stabilised by the active site environment, particularly a tryptophan residue which stacks over the cofactor site. The structure of a galactose oxidase variant in which the tryptophan is mutated to a glycine has been determined and characterised. Past attempts to obtain an enzyme-substrate complex with sugar molecules have been unsuccessful since space at the active site of the original crystal form was limited. Additionally crystals have not been activated prior to substrate incubation. Investigation of substrate binding using activated crystals and a different crystal form have been carried out. The C383S variant protein, has been shown to have improved activity against galactose, however, the wide substrate specificity of the enzyme has allowed investigation of substrate binding using a smaller substrate. Two enzyme-substrate structures, determined to 1.8 A and 1.9 A resolution have be obtained following incubation of C383S with a smaller alcohol substrate. Investigation of the fully reduced form of galactose oxidase has proved difficult due to the high reactivity of Cu'+ with oxygen. Apo-galactose oxidase crystals were anaerobically incubated with Cu" and the structures determined in order to characterise the fully reduced form of galactose oxidase.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.485759
Depositing User: Ethos Import
Date Deposited: 26 Mar 2010 16:43
Last Modified: 07 Mar 2014 11:21
URI: http://etheses.whiterose.ac.uk/id/eprint/649

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