Abstract
Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP–driven production of light. We have used time–resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC–MSMS revealed a similar conformation–dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.
Highlights
It remains a challenge to understand how the function of a protein can be affected when a low affinity drug such as a general anesthetic binds to it
An allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output
Consistent with our observation that TFD-benzyl alcohol photolabeled both Ile-314 and Ser-316, we found that double labeling predominated over single labeling by fourfold with +adenosine 59-triphosphate (ATP) at 200 ms, but the quality of these double labeled spectra were inadequate to resolve their sites of photoincorporation
Summary
It remains a challenge to understand how the function of a protein can be affected when a low affinity drug such as a general anesthetic binds to it. A suitable model is luciferase because it is inhibited by a fairly wide range of general anesthetics and has long been a focus of mechanistic studies. It is readily available and its activity is monitored by its light output [5,6,7,8,9]. A photon is emitted during relaxation of the lactone from the excited state to form oxyluciferin [11]
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