Photoaffinity labeling of the catalytic site of prenyltransferase.

摘要: Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl and 3-azido-1-butyl have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due relatively poor affinity pyrophosphate for enzyme, only (aryl azide) was utilized photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In absence activating light, binding studies demonstrate that o-aryl competes with both natural substrates, isopentenyl geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme irradiated in presence compared irradiation azide, protein loses its capacity direct proportion photolabeling. A stoichiometry 2 mol covalently bound per dimer established [1-3H]-o-azidophenethyl Since there are two sites dimer, appears specifically sites. Additional evidence reagent specific came from observation farnesyl afforded complete protection against photoinactivation, while provided partial protection. Gel isoelectric focusing verified this indicated labeled more acidic point native enzyme.