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|Title:||Some studies on shikimate kinase.|
|Authors:||Buttrum, Mark Eric.|
|Presented at:||University of Leicester|
|Abstract:||The mechanisms of the enzymes of the shikimate pathway, and the mechanisms of phosphoryl transfer by kinases in general are reviewed in chapter 1. In chapter 2, the preparation of B,y-[18O]ATP, alpha,beta-[18O]ADP, alpha,beta-[18O]ATP, and alpha1,beta-[18O]Ap3A are reported. [18O]ATP is used to investigate the mechanism of phosphoryl transfer by shikimate kinase. In the presence of shikimate kinase and shikimate, randomisation of the -18O label with the O2 occurs as expected. No significant randomisation was observed in the presence of a novel fluoro shikimate analogue or in the absence of any cosubstrate. An associative Sn2(P) mechanism of phosphoryl transfer is thereby assumed. In chapter 3, the design, synthesis, and characterisation of analogues of shikimate are reported. An epoxy-shikimate analogue is observed to be a weak irreversible inhibitor of shikimate kinase. Substrate protection studies suggest that the modification is most likely at the ATP binding site. Cis-diol and trans-diol shikimate analogues, lacking the C-5 hydroxyl group, are observed to be substrates of the enzyme. The presence of the C-5 hydroxyl group is found to be important for specificity with a maximum contribution of 3.9 kcal mol-1 to binding energy. A fluoro shikimate analogue, also lacking the C-5 hydroxyl, is found to be a competitive inhibitor, with a Ki of 21 mM. The design, synthesis and characterisation of shikimate analogues with altered hydroxyl stereochemistry at C-3 and C-4, prepared from (5S,6R)-5,6-dihydroxy-l-(trifluoromethyl)-cyclohexa-1,3-diene are reported in chapter 4. Each of the analogues demonstrates similar properties, showing weak non-competitive inhibition with shikimate kinase. The importance of the carboxylate group for the recognition of shikimate is discussed. In chapter 5, shikimate kinase was observed to be inactivated by the reagents iodoacetamide, N-ethyl maleimide, iodoacetate, 2,3 butanedione and diethylpyrocarbonate at pH 7.0. N-ethyl maleimide was the most potent inhibitor, being 60- to 6000-times more potent than the other reagents. The presence of ATP was observed to give total protection against modification by iodoacetamide, N-ethyl pyrocarbonate, and diethylpyrocarbonate, suggesting modification at the active site of the enzyme. Shikimate was found also to partially protect against inactivation by each of these three reagents. A cysteine residue within the probable ATP binding site is tentatively assigned as the site of modification, and its possible roles discussed.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Chemistry|
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