Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents and by chemical modification

Previously we have established that an essential cysteinyl group on the [alpha] subunit is at the aldehyde site of ([alpha beta]) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the essential cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the essential cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. [...]