Chemical modification of bacterial luciferase with ethoxyformic anhydride: evidence for an essential histidyl residue.
Bacterial luciferase is a heteropolymeric protein (alphabeta) that catalyses the conversion of chemical energy to light by oxidation of a reduced flavin mononucleotide and a long chain aliphatic aldehyde. Elucidation of the specific amino acid residues involved in the enzymatic reaction is essential for understanding the mechanisms of the bioluminescent reaction. Luciferase has been found to be inactivated by ethoxyformic anhydride with a second-order rate constant of 146 M-1 min-1 at pH 6.1 and 0 degrees C with a concomitant increase in absorbance at 240 nm due to formation of ethoxyformylhistidyl derivatives. Activity could be restored by hydroxylamine and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Both substrates, FMNH2 and aldehyde, protected the enzyme against inactivation, suggesting that the modification occurred at or near the active site. Incorporation of [14C]ethoxyformyl groups in luciferase indicated that inactivation resulted from the modification of about three histidyl residues, one histidine being found on the alpha subunit and two on the beta subunit. Hybridization experiments, in which ethoxyformylluciferase, alphambetam, was complemented with native subunits, alpha or beta, showed that the hybrid alphambetam, has the same activity as alphambetam whereas the activity of the hybrid alphabetam, was close to that of the reconstituted luciferase alphabeta. The results indicate that modification of only one histidyl residue on the alpha subunit inactivates luciferase and suggest that this histidyl residue plays an essential role in the mechanism of the bacterial bioluminescent reaction.
D000447:Aldehydes / Q000494:pharmacology; D000812:Anhydrides / Q000494:pharmacology; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D005019:Ethyl Ethers / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000494:pharmacology; D006639:Histidine; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D000447:Aldehydes; D000812:Anhydrides; D005019:Ethyl Ethers; D006639:Histidine; D005486:Flavin Mononucleotide; D008156:Luciferases