Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine.
Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.
Rutherfurd
K J
KJ
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Chen
S A
SA
Shively
J E
JE
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000311:Adrenal Glands / Q000187:drug effects / Q000201:enzymology*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000302:isolation & purification; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000345:Affinity Labels; D013438:Sulfhydryl Compounds; D009243:NAD; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D015096:3-Hydroxysteroid Dehydrogenases; D013251:Steroid Isomerases; D000241:Adenosine; D003545:Cysteine; D004229:Dithiothreitol