IDENTIFICATION AND CHARACTERIZATION OF A CATALYTIC BASE IN BACTERIAL LUCIFERASE BY CHEMICAL RESCUE OF A DARK MUTANT

Mutation of the His44 residue of the alpha subunit of Vibrio harveyi l uciferase to an alanine was known to reduce the enzyme bioluminescence activity by five orders of magnitude [Xin, X., Xi, L., and Tu, S.-C. (1991) Biochemistry 30, 11255-11262], We found that the residual activ ity of the alpha H44A luciferase was markedly enhanced by exogenously added imidazole and other simple amines. The peak luminescence intensi ty in nonturnover assays was linearly proportional to levels of alpha H44A and the rescue agent, indicating a lack of significant binding un der our experimental conditions. The rescue effect of imidazole was pH dependent and quantitatively correlated well with the amount of imida zole base. The rescue efficiencies of imidazole and amines were found to be regulated by both their molecular volume and pK(a). A Bronsted a nalysis revealed a beta value of 0.8 +/- 0.1, The enhancement of alpha H44A activity by imidazole took place after the formation of the flav in 4a-hydroperoxide intermediate. The predominant form of the flavin 4 a-hydroperoxide intermediate generated by alpha H44A was inactive in b ioluminescence, but was reactive with the aldehyde substrate for biolu minescence in the presence of imidazole. These findings, taken togethe r, provide evidence for assigning a role for the alpha His44 imidazole as a catalytic base in the luciferase reaction, This study provides t he first characterization of a catalytic residue for bacterial lucifer ase and the first demonstration of the rescue of a histidine-mutated e nzyme by exogenous imidazole and amines.