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|Title:||Spectroscopic studies of chloramphenicol aceyltransferase.|
|Presented at:||University of Leicester|
|Abstract:||Chloramphenicol acetyltransferase (CAT) is the enzyme responsible for the high level resistance to chloramphenicol exhibited by many prokaryotes. The enzyme operates by way of 0-acetylation of the primary (C-3) hydroxyl of chloramphenicol using acetyl coenzyme A as the acyl donor. The primary aim of the investigations described in the thesis was to characterise the intrinsic fluorescence properties of CAT(III). It has been possible, by site-directed mutagenesis experiments performed by D.I.A. Murray, to determine the contribution of each of the three tryptophan residues of CAT(III) to its intrinsic fluorescence, and the response of the latter to the binding of substrates. Of the three tryptophans, only Trp 152 is both absolutely conserved and in close contact with one of the substrates (CoA). Fluorescence enhancement on the binding of acetyl CoA is largely due to Trp 152, whereas the binding of chloramphenicol can be quantitated and monitored via a decrease in the fluorescence contributions from both Trp 86 and Trp 152. The fluorescence responses of wild type CAT(III) allowed the development of an independent spectroscopic method for the study of the formation of each of the binary complexes of CAT and its substrates. The binding affinities obtained in this manner were in agreement with data from steady state and equilibrium dialysis. Substrate synergism in the system was studied by way of non-productive ternary complexes and was demonstrated to be dependant on the interaction of substrates with the proposed catalytic residue, His 195. The availability of an independent spectroscopic binding assay has provided the opportunity to study CAT by transient kinetic methods. Stopped-Mow fluorimetry techniques have allowed the measurement of the association and dissociation rate constants for chloramphenicol.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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