Computational and experimental studies of interactions between general anesthetics and proteins.

Abstract

The molecular mechanism of general anesthesia remains unknown. Potential anesthetic target sites include membrane lipid, protein or possibly both. Computational and experimental studies were done to determine if the functions of isolated protein systems can be effected by anesthetic. A total of six enzymes: bacterial luciferase, acetylcholinesterase, papain, chymotrypsin, hexokinase and glyceraldehyde-3-phosphate dehydrogenase were evaluated for potential anesthetic sensitivity. For the computational studies on bacterial luciferase (V. Harveyi), an optimized structure of the α subunit provided a basis for performing Hyperchem electrostatic potential calculations. A total of 11 anesthetic and 1 convulsant model were used in constructing electrostatic contour plots of the active site. All anesthetics substantially decreased the field gradient at the active site, while the convulsant increased the gradient. For the computational studies on the remaining five enzymes, protein optimizations and docking analyses were performed with the INSIGHTII program. Models for halothane, methoxyflurane and nitrous oxide were built and docked into enzyme active sites. Intermolecular energy data revealed two possible anesthetic sensitive proteins: papain and, to a lesser degree, glyceraldehyde-3-phosphate dehydrogenase. Experimental enzyme assays for the five enzymes were undertaken to determine activity in the presence and absence of halothane. Activities were measured spectrophotometrically over a wide range of substrate concentrations. Substrate concentrations both spanned and exceeded the Km value of the enzyme under analysis. Substrate-activity curves were constructed to evaluate competitive effects on enzyme kinetics (increased Km). Competitive effects on enzyme kinetics were not found for acetylcholinesterase, α-chymotrypsin and hexokinase. A slight but probably not significant effect of halothane on glyceraldehyde-3-phosphate dehydrogenase activity was observed, as shown by the small increase in Km and the tabulated %control values. Papain was found to be inhibited by nearly 50% at saturating substrate concentrations and by approximately one-third at lower substrate concentrations. From these theoretical and experimental studies on anesthetic-protein interactions, two isolated protein systems (bacterial luciferase and papain) were observed to display an anesthetic sensitivity. These studies have served to support the theory that proteins are plausible sites of anesthetic action and thus should be studied as potential anesthetic target sites.