Abstract
Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.
Highlights
From the Departmentof Pharmacology, University of Pennsylvania School of Medicine and the §Departmentof Anatomy and Histology, liniversity of Pennsyluania School of Dental Medicine, Philadelphia, Pennsyluania 19104
Completeacid functions asa quinone reductase (2), an aromatailccohol hydrolysis of 3a-HSD radiolabeled with either agent dehydrogenase (a),a dihydrodiol dehydrogenase (2,4,5), and followed by amino acid analysis led to the identifica- a hydroxyprostaglandin dehydrogenase (6)
[’“CIBrAndand [‘“CIBrllPcovalently tag discrete re- ductase which is important in drug and xenobiotic metaboactive cysteine(s)at theenzyme active site
Summary
Of 30-HSD radiolabeled with [14C]BrAndfollowed by peptide 3A1 was sequenced onlya single PTH-derivative was released mapping via ion pair RP-HPLCgave a peptide map in which per cycle yielding unambiguous sequence data. Cysteine came from aligning the peptide sequence with the The labeled active site peptide (3A1) gave an amino acid nucleotide sequence of the cDNA for 3a-HSD (Fig. 6). This composition consistent with a decapeptide containing a car- alignment clearly shows that the peptidesequenced contains boxymethylcysteine residue: Asx~,CMC,, Serp, Glyl, Arg,, only one cysteine and that it is present atposition 9 of this Vall, Ilel, and Phel. Peptide l l P 3 gave data consistent with the following amino acid composition (numbers in parentheses indicate amaincoid molar ratio): Asx (2.0), Ser (l.O), Ala (2.0), Pro (2.0), Val (1.8), Ile ( L O ) , Leu (2.5), Lys (1.6), and ['4C]carboxymethylcysteine was identified in the effluent of the amino acid analysis column. Carboxymethylcysteine was assigned to position 11of l l P 3 since this amino acid was not recovered quantitatively in the sequence
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