Chemical modification and characterization of the alpha cysteine 106 at the Vibrio harveyi luciferase active center.
Vibrio harveyi luciferase, an alpha beta dimer, was effectively inactivated by treatment with the methylation agent methyl p-nitrobenzene sulfonate. However, inactivation of luciferase in the presence of excess amounts of this reagent did not follow pseudo-first-order kinetics. After taking the autodecay of this reagent into consideration in kinetic analysis, the pseudo-first-order constants and subsequently the second-order rate constant (83 min-1 M-1 at pH 7 and 23 degrees C) were determined. The inactivation rate can be retarded by the addition of the decanal or the reduced FMN substrate but not by the reaction product FMN. The binding of decanal specifically protected one target residue against modification with a concomitant protection of luciferase against inactivation. A pentapeptide containing this specific target residue was isolated and identified to be Phe-Gly-Ile-X-Arg with X corresponding to the S-methylated form of the cysteinyl residue at position 106 of the luciferase alpha subunit. It is concluded that this reactive alpha Cys-106 is at the aldehyde site and is also near the reduced flavin site of luciferase. The modified enzyme exhibited no gross conformational changes detectable by protein fluorescence measurements, which may be due to the small size change of the target cysteinyl residue after methylation. The methylated enzyme still retained the ability to bind one decanal and one reduced FMN without any substantial changes in binding affinities. The cause of luciferase inactivation by the methylation of alpha Cys-106 has been shown to be the impaired ability to form the 4a-hydroperoxy-flavin intermediate from the bound flavin substrate or to stabilize this intermediate.
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D000595:Amino Acid Sequence; D001557:Benzenesulfonates / Q000494:pharmacology*; D001665:Binding Sites; D003545:Cysteine; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D014733:Vibrio / Q000201:enzymology*
D001557:Benzenesulfonates; D005415:Flavins; C010233:methyl-4-nitrobenzenesulfonate; D008156:Luciferases; D003545:Cysteine