Affinity labeling of the aldehyde site of bacterial luciferase.
2-Bromo[1-14C]1-decanal was synthesized as an affinity labeling probe for the aliphatic aldehyde site of Vibrio harveyi luciferase. In the presence of excess amounts of this probe, the inactivation of bacterial luciferase occurred following apparent first order kinetics. This inactivation was markedly retarded in the presence of decanal but neither butanal (a very poor aldehyde substrate) nor FMN (a reaction product derived from reduced FMN) showed any significant protective effect. Upon mixing luciferase with the affinity labeling probe, a noncovalent complex was formed prior to the covalent attachment. At pH 6 and 23 degrees C, the dissociation constant for the binding step and the rate constant for the covalent modification step were determined to be 23 microM and 1 min-1, respectively. The displacement of a bound aldehyde substrate by this probe added secondarily was also demonstrated. The inactivation of luciferase was correlated with both the incorporation of about 1.2 molecules of the probe and the loss of 0.8 to 1.1 cysteinyl residues/luciferase alpha beta dimer. The presence of an essential sulfhydryl group at the aldehyde site of luciferase has thus been demonstrated. This sulfhydryl group was a constituent residue of the alpha subunit and was near the alpha beta subunit interface. This residue appears to be the same essential cysteinyl group previously identified by chemical modification (Nicoli, M.Z., Meighen, E.A., and Hastings, J.W. (1974) J. Biol. Chem. 249, 2385-2392). The labeled luciferase did not exhibit any significant binding for the reduced FMN substrate.
D000345:Affinity Labels / Q000494:pharmacology*; D000447:Aldehydes / Q000494:pharmacology*; D005486:Flavin Mononucleotide / Q000032:analysis; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry; D011485:Protein Binding; D014733:Vibrio / Q000201:enzymology*
D000345:Affinity Labels; D000447:Aldehydes; D005486:Flavin Mononucleotide; C042471:2-bromo-1-decanal; D008156:Luciferases