Affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside analog.
Incubation of 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD; E.C.1.1.1.53) with the nucleoside 5'-p-fluorosulfonylbenzoyladenosine (FSA) caused a time-dependent and irreversible loss in enzyme activity. Both 3 alpha- and 20 beta-hydroxysteroid oxidoreductase activities decreased at equal rates by a first order kinetic process (in 0.05M phosphate buffer at pH 6.0 and 25 degrees C, t1/2 = 170 min). Incubation of 3 alpha, 20 beta-HSD was quenched by addition of 2-mercaptoethanol which instantaneously reacts with the fluorosulfonyl group of FSA. The cofactor NADH protected 3 alpha, 20 beta-HSD against inactivation by FSA, in a concentration-dependent manner. However, progesterone did not protect 3 alpha, 20 beta-HSD against inactivation by FSA. Evidently, FSA causes inactivation of the enzyme by irreversibly binding to the NADH-binding region at the active site of 3 alpha, 20 beta-HSD. Both 3 alpha- and 20 beta-hydroxysteroid oxidoreductase activities disappeared at equal rates under a variety of enzyme-inactivating conditions. These results suggest that both 3 alpha- and 20 beta-activities occur at the same active site of 3 alpha, 20 beta-HSD.
D015089:20-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D000241:Adenosine / Q000031:analogs & derivatives*; D000345:Affinity Labels; D055598:Chemical Phenomena; D002621:Chemistry; D003349:Cortisone Reductase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009243:NAD; D013302:Streptomyces / Q000201:enzymology*
D000345:Affinity Labels; D009243:NAD; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D015089:20-Hydroxysteroid Dehydrogenases; D003349:Cortisone Reductase; D000241:Adenosine