Affinity labeling of the active site of pig liver NADH-cytochrome b5 reductase by 5′-p-fluorosulfonylbenzoyladenosine
Tóm tắt
Lysosome-solubilized pig liver NADH-cytochrome b5 reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/Kobs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.Kdor the initial reversible enzyme-analogue complex is estimated at 185 µM withK2=0.22 min−1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[14C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b5 reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.
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