Bacterial Luciferase

Abstract

Abstract Bacterial luciferase is inactivated by modification of a single reactive sulfhydryl group on the α subunit by alkylating reagents. The apparent second order rate constants for inactivation by N-alkylmaleimides increase markedly with increasing chain length of the alkyl substituent, from 2 to 8 carbon atoms, suggesting noncovalent binding of the alkyl side chains mediated by hydrophobic interactions with the enzyme. The change in free energy of binding is about -0.5 Cal per mole per additional methylene group. The reactive sulfhydryl group thus must be in or near a hydrophobic region of the enzyme. At high maleimide concentrations, the rate of inactivation approaches a concentration-independent maximum (60 min-1, 0.02 m phosphate, pH 7.0, 25°) which is independent of alkyl chain length. The proposed candidates for the emitting species in the bioluminescence reaction all require highly nonpolar environments to blue-shift their fluorescence maxima to match that of the bioluminescence. These results indicate that such an environment is indeed available in the active center of bacterial luciferase.