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 luciferase 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 activity of the alpha H44A luciferase was markedly enhanced by exogenously added imidazole and other simple amines. The peak luminescence intensity in nonturnover assays was linearly proportional to levels of alpha H44A and the rescue agent, indicating a lack of significant binding under our experimental conditions. The rescue effect of imidazole was pH dependent and quantitatively correlated well with the amount of imidazole base. The rescue efficiencies of imidazole and amines were found to be regulated by both their molecular volume and pKa. A Brønsted analysis revealed a beta value of 0.8 +/- 0.1. The enhancement of alpha H44A activity by imidazole took place after the formation of the flavin 4a-hydroperoxide intermediate. The predominant form of the flavin 4a-hydroperoxide intermediate generated by alpha H44A was inactive in bioluminescence, but was reactive with the aldehyde substrate for bioluminescence in the presence of imidazole. These findings, taken together, provide evidence for assigning a role for the alpha His44 imidazole as a catalytic base in the luciferase reaction. This study provides the first characterization of a catalytic residue for bacterial luciferase and the first demonstration of the rescue of a histidine-mutated enzyme by exogenous imidazole and amines.
Huang
S
S
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934, USA.
Tu
S C
SC
D000588:Amines / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003624:Darkness; D005415:Flavins / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010545:Peroxides / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology
D000588:Amines; D001426:Bacterial Proteins; D005415:Flavins; D007093:Imidazoles; D008164:Luminescent Proteins; D010545:Peroxides; D006639:Histidine; D008156:Luciferases