Chemical modification of bacterial luciferase with ethoxyformic anhydride: evidence for an essential histidyl residue

Abstract

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.

Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

“…The His-modifying agent DEPC is commonly used to assess possible involvement of His residues in native or recombinant ion channel or transporter function and, in particular, their involvement in pH sensitivity. DEPC carboxyethylates the proton-titratable imidazole group of His in a pH-sensitive reaction (7,29), and DEPC specificity for His modification has been claimed at pH 5.5-7.5 (3). We have shown that DEPC can reversibly inhibit AE2-mediated Cl Ϫ transport in Xenopus oocytes, in contrast to its irreversible inhibition of kAE1 ( Fig.…”

Histidyl residues at the active site of the Na/succinate cotransporter in rabbit renal brush borders

“…At higher concentrations of DEP, a second irreversible carbethoxylation may take place (step 2). Hydroxylamine will catalyze an opening of the dicarbethoxylated histidyl ring (step 3) tion process although generally very fast, sensitive, and complete (Burstein et al, 1974;Cousineau & Meighen, 1976) can be very slow (Miles & Kumagai, 1974;Wiejnans & Muller, 1982). Another explanation for the lack of complete decarbethoxylation by NH2OH is the possible irreversible carbethoxylation of the second nitrogen atom in the imidazole ring followed by an opening of the ring in the presence of NH2OH (see Fig.…”

Interactions of Flavins With Amino Acid Residues: Assessments From Spectral and Photochemical Studies

“…Some specify binding of substrate, e.g. His in pyridoxamine phosphate oxidase (Tsuge and McCormick, 1977), and in a bacterial luciferase, where FMNH, is a cosubstrate (Cousineau and Meighen, 1976). In some instances, it is not clear what the role of a given residue may be, even when the proximity to flavin may contribute to observed fluorescence quenching, e.g.…”