A photoaffinity probe 1-diazo-2-oxoundecane has been synthesized and used to examine the aldehyde-binding site of the nonidentical dimeric luciferase (αβ) from Vibrio harveyi cells. In the dark, the probe competes against aldehyde in binding to luciferase. Irradiation of luciferase and the probe at 254 nm resulted in primarily specific labeling of both α and β subunits with concomitant enzyme inactivation, but significant (≃40%) nonspecific labeling of mainly the β subunit also occurred. The addition of decanal to protect the active center reduced the rate of inactivation. When 2-mercaptoethanol was included to quench the nonspecific labeling, the amounts of probe incorporated into α and β correlated stoichiometrically with the quantities of enzyme photoinactivated. On the basis of these findings, we postulate that the aldehyde binding site is at or near the subunit interface of luciferase.
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