The anaesthetic sensitivity of a highly purified luciferase enzyme from V ib rio
h arveyi luminous bacteria has been assessed in detail and compared with the
corresponding pharmacological profile of whole animals. Of the fifty—four different
anaesthetics tested, almost all were found to inhibit bacterial luciferase by preventing
the binding of the long—chain aldehyde substrate. Differences in the details of the
kinetics of reactions inhibited by different groups of anaesthetics were observed; these
were investigated and accounted for. Significantly, the potency of n —alcohols and
n —alkanes as luciferase inhibitors increases with chain—length but then, quite
suddenly, disappears. This "cut-off" behaviour, which was attributed to the finite
dimensions of the luciferase pocket, mimics the cu t-off in anaesthetic potencies of
these agents, strongly suggesting that the target sites underlying general anaesthesia
are protein pockets.
Notably, bulky halogenated agents are much less potent as luciferase inhibitors
than as anaesthetics, indicating the comparative narrowness of the enzyme pocket.
To test this hypothesis, the potencies, as inhibitors of luciferase, of cycloalcohols and
n — alcohols — compounds which, though chemically similar, are structurally distinct
— were compared. As predicted, the bulkier cycloalcohols are much less effective,
even when their greater aqueous solubility (measured using a novel experimental
technique) is taken into account. Analysis of alcohol and alkane data showed that
the distribution of polar and apolar regions in the luciferase pocket, as well as its
geometry, distinguishes it from the target sites in animals.
Surprisingly, the anaesthetic sensitivity of V . h arveyi luciferase was found to be
quite distinct from that of V . fisc h e ri luciferase; moreover the NADH:FMN
oxidoreductase of V . harveyi was shown to be inhibited by halothane. The bearing
of these results on previously published findings is discussed.
Measurement of the stabilisation of the luciferase—peroxyflavin complex by
n — alcohols confirmed the luciferase cu t-o ff effect and revealed that this reaction
intermediate has a much higher affinity for long —chain compounds than the enzyme
on its own. This difference in affinities accounts for observed differences in the
kinetics of reactions inhibited by short— and long—chain alcohols and alkanes.