Year: 1989
Type: dissertation
Abstract: The anaesthetic sensitivity of a highly purified luciferase enzyme from V ib rio \nh arveyi luminous bacteria has been assessed in detail and compared with the \ncorresponding pharmacological profile of whole animals. Of the fifty—four different \nanaesthetics tested, almost all were found to inhibit bacterial luciferase by preventing \nthe binding of the long—chain aldehyde substrate. Differences in the details of the \nkinetics of reactions inhibited by different groups of anaesthetics were observed; these \nwere investigated and accounted for. Significantly, the potency of n —alcohols and \nn —alkanes as luciferase inhibitors increases with chain—length but then, quite \nsuddenly, disappears. This "cut-off" behaviour, which was attributed to the finite \ndimensions of the luciferase pocket, mimics the cu t-off in anaesthetic potencies of \nthese agents, strongly suggesting that the target sites underlying general anaesthesia \nare protein pockets. \nNotably, bulky halogenated agents are much less potent as luciferase inhibitors \nthan as anaesthetics, indicating the comparative narrowness of the enzyme pocket. \nTo test this hypothesis, the potencies, as inhibitors of luciferase, of cycloalcohols and \nn — alcohols — compounds which, though chemically similar, are structurally distinct \n— were compared. As predicted, the bulkier cycloalcohols are much less effective, \neven when their greater aqueous solubility (measured using a novel experimental \ntechnique) is taken into account. Analysis of alcohol and alkane data showed that \nthe distribution of polar and apolar regions in the luciferase pocket, as well as its \ngeometry, distinguishes it from the target sites in animals. \nSurprisingly, the anaesthetic sensitivity of V . h arveyi luciferase was found to be \nquite distinct from that of V . fisc h e ri luciferase; moreover the NADH:FMN \noxidoreductase of V . harveyi was shown to be inhibited by halothane. The bearing \nof these results on previously published findings is discussed. \nMeasurement of the stabilisation of the luciferase—peroxyflavin complex by \nn — alcohols confirmed the luciferase cu t-o ff effect and revealed that this reaction \nintermediate has a much higher affinity for long —chain compounds than the enzyme \non its own. This difference in affinities accounts for observed differences in the \nkinetics of reactions inhibited by short— and long—chain alcohols and alkanes.
Source: Spiral (Imperial College London)
Author: Stephen Curry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2762533548