Structural Basis for the Inhibition of Firefly Luciferase by a General Anesthetic

Abstract

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

Binding of the General Anesthetics Propofol and Halothane to Human Serum Albumin

“…All of the binding sites were predominantly apolar, although most also showed evidence of significant polar interactions between charged or polar amino acids and the polarizable halogen atoms, particularly the bromine. The possible importance of polar interactions between proteins and halogenated compounds has been noted before (3,28), and the likelihood that general anesthetic binding sites are amphiphilic in nature has been stressed by our group (29,30) and others (31)(32)(33).…”

“…(14,23). 3 The domains are color-coded as follows: red, domain I; green, domain II; blue, domain III. The A and B sub-domains within each domain are depicted in dark and light shades, respectively.…”

“…Superposition of the fatty acid structures 3 indicates that the binding of this propofol molecule could be prevented by ligands that bind to fatty acid binding site FA5. It is probable that the first of the two propofol binding sites (PR1 in subdomain IIIA) has the highest affinity because, during one experiment where the crystals were partially back-soaked and the propofol concentration was reduced, the electron density for the second propofol molecule PR2 disappeared, whereas that for the first molecule was easily interpretable (data not shown).…”

“…(Although the position of the electron-dense bromine atom was always clear, there was some ambiguity about the relative positions of the chlorine atom and the CF 3 group. In most cases the shape of the density was used to guide positioning of the slightly bulkier CF 3 group, but because the data are limited to 2.4 Å resolution and the model B-factors are relatively high, the orientations modeled cannot be regarded as definitive.) Two of these halothane molecules (HAL1 and HAL2) bind within a solvent-exposed trough at the interface between subdomains IIA and IIB, which can also bind a fatty acid molecule (FA6).…”

“…However, there are several soluble proteins to which anesthetics are known to bind, and studies with these proteins have provided valuable information on the nature of anesthetic binding sites. Most of this work has been done with serum proteins and luciferase enzymes, but so far the only example of an anesthetic-sensitive protein for which there is also high resolution structural data is firefly luciferase (3).…”