Bacterial Luciferase: CHEMISTRY OF THE REACTIVE SULFHYDRYL

Abstract

Abstract Bacterial luciferase catalyzes the bioluminescent oxidation of FMNH2 and a long chain aliphatic aldehyde by O2, producing FMN and the corresponding carboxylic acid. The enzyme from bacterial strain MAV has been found to be inactivated by modification of a single cysteinyl residue per enzyme molecule by alkylating reagents such as iodoacetamide, iodoacetate, and N-ethylmaleimide. The apparent second order rate constants for inactivation at pH 7.0 (25°) are 1.67 x 103 m-1 min-1 for N-ethylmaleimide and 7.0 m-1 min-1 for iodoacetamide. Inactivation by iodoacetate at pH 7.0 is much less rapid, and the rate constant was not determined below pH 8. With all three reagents, the rate of inactivation increases with increasing pH in a manner suggesting a pKa of about 9.4 for the reactive sulfhydryl group. The rate of inactivation by N-[1-14C]ethylmaleimide correlates with the rate of modification of one sulfhydryl group on the α subunit, the β subunit being modified much less rapidly. Long chain aliphatic aldehydes protect the enzyme against inactivation by N-ethylmaleimide, the degree of protection increasing with increasing aldehyde chain length. The reactive sulfhydryl is also protected by FMN against alkylation. No binding of FMNH2 could be measured for iodoacetamide- or N-ethylmaleimide-modified enzyme. These results suggest that the reactive sulfhydryl is in or near the active center of the enzyme. The pH profiles of FMNH2 binding affinity and several catalytic parameters of the native enzyme do not implicate a group with a pKa near that of the sulfhydryl. The catalytic function (if any) for the reactive cysteine remains unknown.