Human Placental Enzyme Research Articles

Synthesis of steroidal [4,6-b,c]-benzothiazepines, a new class of aromatase inhibitor

Several steroidal [4,6-b,c]benzothiazepines have been prepared via base-catalyzed Michael addition of 2-aminothiophenol to 3β,17β-diacetoxyandrost-4-en-6-one. The product of this reaction has the 4β,5α stereochemistry, but cyclizes to a benzothiazepine with the steroidal 4β,5β configuration, confirmed by X-ray crystallographic analysis. 2-Aminothiophenol reacts with 3β,17β-diacetoxyandrost-4-en-6-one under acidic conditions to give a steroidal 6-spiro-benzothiazole. An androst-4-ene-3,17-dione-based [4,6]benzothiazepine has been shown to be a moderate competitive inhibitor of the human placental aromatase enzyme with IC50 = 42.3 µM. Keywords: steroid, aromatase, benzothiazepine.

Diadenosine 5′,5‴- P1, P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets

An asymmetrically-cleaving diadenosine 5′,5‴- P 1, P 4-tetraphosphate hydrolase (Ap 4A → ATP + AMP) is present in all higher eukaryotes and contributes to the regulation of the intracellular level of the alarmone nucleotide diadenosine 5′,5‴- P 1, P 4-tetraphosphate (Ap 4A). This enzyme has previously been isolated from unfractionated human blood cells. The aim of this report is to determine the contribution made by different blood cell types as part of our study of the roles of Ap 4A as an intra- and extracellular signalling molecule. Ap 4A hydrolase was partially purified from isolated human erythrocytes, leukocytes and platelets by high performance gel permeation chromatography and characterized by kinetic analysis and by probing immunoblots with an antibody raised against the human placental enzyme. Ap 4A hydrolase was clearly present in all three cell types. Each enzyme comprised a single polypeptide of M r 19,200. The erythrocyte and platelet enzymes had a K m for Ap 4A of 0.70 ± 0.05 μM ( n = 3) while the K m for the leukocyte enzyme was 1.50 ± 0.20 μM ( n = 3). All three enzymes showed substrate inhibition above 10 μM Ap 4A. The specific activity of the enzyme in erythrocytes was 0.067 U/10 6 cells, 15-fold lower than that in leukocytes and platelets. However, the erythrocyte hydrolase accounted for 97% of the total activity of unfractionated blood cells (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets and erythrocytes all contain Ap 4A hydrolase activity. The last observation is of particular interest given the reported absence of Ap 4A from enucleated erythrocytes. Two possible interpretations are that the enzyme is retained from the reticulocyte stage, or that erythrocytes retain a capacity, previously unobserved, for dinucleoside polyphosphate synthesis.

A single mutation at lysine 426 of human placental S-adenosylhomocysteine hydrolase inactivates the enzyme.

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy), as well as the reverse reaction, through a mechanism involving an NAD(+)-dependent oxidation of the 3'-hydroxyl group of AdoHcy (3'-oxidative activity), followed by elimination of Hcy to form 3'-keto-4',5'-didehydro-5'-deoxy-Ado. The addition of water at the 5'-position (5'-hydrolytic activity) of this tightly bound intermediate, followed by an NADH-dependent reduction, results in the formation of Ado. Based on a computer graphics model of the active site of this enzyme, it was hypothesized that amino acid residues at the carboxyl-terminal end of the protein reside in the active site of the enzyme and could play a role in catalyzing the 5'-hydrolytic reaction (Yeh, J. C., Borchardt, R. T., and Vedani, A. (1991) J. Comput. Aided Mol. Des. 5, 213-234). Using site-directed mutagenesis, we show here that lysine 426 is essential for the catalytic activity of the enzyme and that it appears to play a crucial role in the 5'-hydrolytic activity and/or stability of the quaternary structure of the human placental enzyme. Mutation of Lys-426 to arginine (K426R) produces a stable tetrameric enzyme that lacks overall catalytic activity and that was isolated predominantly as its NADH form containing tightly bound 3'-keto-Ado, suggesting that the K426R mutant has oxidative activity, but lacks 5'-hydrolytic activity, preventing it from completing the entire catalytic cycle. Mutations of Lys-426 to glutamic acid (K426E) and alanine (K426A) produce enzymes that exist primarily as monomers, do not bind NAD+ or NADH, and lack catalytic activity. The results of the Lys-426 mutations suggest that this lysine residue is crucial for the 5'-hydrolytic activity of the enzyme and/or stabilizing the quaternary structure of the enzyme.

Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD + -dependent 15-hydroxyprostaglandin dehydrogenase

NAD +-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this study the cDNA for human placental 15-PGDH was expressed in Escherichia coli and che recombinant enzyme was purified to homogeneity and characterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. Determinations of K m and V ax values for a number of the prostaglandins and NAD + indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directed mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are known to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. Western blot analysis indicated that the mutant and wild-type proteins were expressed at the similar levels. However, all of the mutant proteins were found to be inactive. These results indicate that both tyrosine-151 and lysine-155 are required for 15-PGDH activity.

Lack of inhibition of placental estrone sulfatase and aromatase enzymes by vitamin D 3 and its analogs

The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 1α25-dihydroxyvitamin D 3 and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D 3 and vitamin D 3 sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 μm. Only vitamin D 3 sulfate and 1α25-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 μm each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer cells in vivo.

Original ArticlesExpression of Engineered Human I7β-Estradiol Dehydrogenase in a Prokaryotic System

17 beta estradiol dehydrogenase (17 beta DH) is a model for pyridine-dependent steroid-converting enzymes. To define the structural and functional parameters of 17 beta DH, we created an expression system for production of abundant, homogeneous enzyme. A full-length 17 beta DH cDNA clone was engineered into the inducible expression vector pMON 5839. After induction of plasmid-bearing Escherichia coli JM109 cells, the authenticity of the recombinant human placental 17 beta DH (r17 beta DH) was evaluated. Protein electrophoresis and Western blot analysis confirmed the immunologic identity of r17 beta DH with native human placental enzyme. The amino acid sequence, enzyme activity, Vmax, K(m), and kcat of r17 beta DH matched that of the native enzyme. Prokaryotic cell lines offer the opportunity to create an unlimited supply of recombinant human placental 17 beta DH without the expense and time commitment of baculoviral or eukaryotic cell lines. We are now able to use r17 beta DH and its mutants to elucidate the mechanisms of action of this class of enzyme.

Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

Expression of 17β-hydroxysteroid dehydrogenase in the rat ovary during follicular development and luteinization induced with pregnant mare serum gonadotrophin and human chorionic gonadotrophin

Antibodies against human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) and 17-HSD cDNA were used to study the expression of the corresponding enzyme in the immature rat ovary during follicular development and luteinization, which were induced by treating the animals with pregnant mare serum gonadotrophin (PMSG) or with PMSG followed by human chorionic gonadotrophin (hCG). Immuno-blot analysis indicated that the M(r) of the 17-HSD expressed in rat granulosa cells was 35,000, as previously shown for the human placental enzyme. In immunohistochemical studies of untreated immature rat ovaries, only the granulosa cells from small antral follicles were stained. One day after PMSG treatment, strong expression of 17-HSD was observed in the granulosa cells of growing Graafian follicles. A marked decrease in enzyme expression was observed in preovulatory follicles on day 2 of PMSG treatment, starting from the basal layers of granulosa cells and progressing toward the luminal cells. No 17-HSD expression was detected in luteinized follicles or corpora lutea 22 h after hCG injection. The stroma and theca cells were negative for 17-HSD staining. In Northern hybridization analyses, two 17-HSD mRNAs were detected (1.4 and 1.7 kb). The strongest expression for both mRNAs was detected after 1 day of PMSG treatment, coinciding with maximal immunostaining of the enzyme protein. Down-regulation of 17-HSD observed by immunohistochemistry was reflected in a similar decrease in mRNA expression and the signals were almost undetectable 22 h after hCG injection. Our data suggest that 17-HSD expression in rat granulosa cells is up-regulated during follicular development and, thereafter, the enzyme expression is down-regulated during luteinization.

Substituted 1-[(benzofuran-2-YL)-phenylmethyl]-imidazoles as potent inhibitors of aromatase in vitro and in female rats in vivo

Substituted 1-[(benzofuran-2-yl)-phenylmethyl]-imidazoles are a new class of potent aromatase inhibitor with in vitro IC 50 values < 10 nM for certain members using human placental enzyme. At a dose of 2 mg/kg in PMSG-stimulated rats, selected compounds effectively reduce the oestradiol levels by 82–98%.

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.

Studies on cytosolic guanylate cyclase from human placenta

We have purified the soluble form of guanylate cyclase from human placenta >2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 ± 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.

Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262.

Human aldose reductase (EC 1.1.1.21) and aldehyde reductase (EC 1.1.1.2) are implicated in the development of diabetic complications by a variety of mechanisms, and a number of drugs to inhibit these enzymes have been proposed for the therapy and prevention of these complications. To probe the structure and function of these two enzymes, we used site-directed mutagenesis in the cDNAs of both enzymes to replace lysine 262 with methionine. Wild-type and mutant enzymes were overexpressed in Escherichia coli and purified by anion exchange and affinity chromatography. N-terminal sequence analysis, Western blots, and kinetic studies confirmed the identity of the recombinant wild-type enzymes with the native human placental and liver enzymes. Recombinant aldose reductase (hAR) and aldehyde reductase (hGR) have apparent kinetic constants virtually identical to their respective native enzymes. The mutant aldose reductase (hARK262 greater than M) shows a 66-fold increase in Km for NADPH with respect to the wild type (1.9 +/- 0.4 microM versus 125 +/- 14 microM), whereas the Km for DL-glyceraldehyde increased 35-fold (20 +/- 2 versus 693 +/- 41 microM). The same constants for the mutant aldehyde reductase (hGRK262 greater than M) increased 97- and 86-fold, respectively (from 2.0 +/- 0.4 to 194 +/- 16 microM and from 1.6 +/- 0.4 to 137 +/- 3 mM). These results indicate that lysine 262 in aldose reductase and aldehyde reductase is crucial to their catalytic activity by affecting co-factor binding.

Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein

Estrogen synthetase (aromatase) is a cytochrome P-450 enzyme system which converts androgens to estrogens. Although this enzyme has been purified and the cDNA-derived amino acid sequence elucidated, very little is known regarding post-translational modifications of this physiologically crucial enzyme. We show here that the cytochrome P-450 component, P-450 ES, purified from human term placental microsomes, is a glycoprotein based on the following evidence: its molecular weight is decreased following treatment with endoglycosidase F, concanavalin A-biotin specifically binds to this protein immobilized on nitrocellulose, and its oligosaccharide composition is consistent with a single N-linked fucosylated complex type carbohydrate chain. In a reconstitution system, the aromatase activity using the endoglycosidase F-treated P-450 ES was reduced by about 35–40% relative to the native form, regardless of whether androstenedione or testosterone was used as substrate.

The basic isoelectric form of α- l-fucosidase from the hepatopancreas of the shrimp Penaeus monodon (crustacea: decapoda)

1. 1. α- l-Fucosidase was purified ca 10,889-fold to homogeneity from Penaeus monodon, with a final spec. act. of 31,250 U/mg of protein. 2. 2. By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp α- l-fucosidase were discovered to have mol. wts of 63,000 and those of human placental enzyme, 46,000 and 20,000. Since the active shrimp α- l-fucosidase was found to have a mol. wt of 233,000 by Superose ™ 12 FPLC, it was concluded that the purified shrimp enzyme was tetrameric. 3. 3. In contrast to the discovery of thermolability with human placental α- l-fucosidase, the shrimp enzyme was found to be stable to heating at 65°C for 10 min. 4. 4. The shrimp α- l-fucosidase has an isoelectric point (pI) of 8.5, but the human placental enzyme has a pI of 4.0. The shrimp enzyme was sialyated. 5. 5. The shrimp α- l-fucosidase has a pH optimum at 5.5 and its K m was 22.2 μM with 4-methyl-umbelliferyl-α- l-fucopyranoside as substrate. The human enzyme has a broad pH optimum between 5.0 and 6.5.

Purification and properties of a beta-mannosidase from shrimp ( Penaeus japonicus) hepatopancreas

1. 1. A β-mannosidase was purified ca 720-fold to homogeneity from Penaeus japonicus, with a final spec. act. of 252 U/mg of protein. 2. 2. By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp β-mannosidase were discovered to have mol. wts of 31,000 and those of human placental enzyme have similar mol. wts. 3. 3. The shrimp β-mannosidase has an isoelectric point (pI) of 5.6 ± 0.1, and the human placental enzyme has an identical pI. Both enzymes were sialyated. 4. 4. The shrimp β-mannosidase has a pH optimum at 5.0 and its K m was 123 μM with 4-methylumbelliferyl-β- d-mannopyranoside as substrate. The human enzyme has pH optimum at 4.5 and its K m was 10 μM. 5. 5. In contrast to the discovery of thermostability with human placental β-mannosidase, the shrimp enzyme was found to be labile to heating at 45°C for 20 min. Both enzyme activities were inhibited by Hg 2+ and Cd 2+ ions. However, the shrimp enzyme is significantly more sensitive to the inhibition.

Kinetic aspects of human placental alkaline phosphatase enzyme membrane

The crosslinking of alkaline phosphatase of human placenta with human serum albumin has been optimized. During the physico-chemical characterization of this immobilized biocatalyst, special attention was paid to attributes such as the irreversibility of the enzyme support bonding, the stability of the catalytic activity, and the effects of pH and temperature on this activity. Regarding stability, patterns of denaturation are proposed, to account for inactivation curves over time and under storage/operation conditions. These patterns, in some cases, indicate the existence of different populations of immobilized enzyme molecules, with a different degree of sensitivity to denaturation. The activity vs pH profiles are clearly modified by the immobilization process. This is because the pH of the free homogeneous solution, measurable with a pH-meter, differs from the real pH of the immediate microenvironment of the immobilized enzyme molecules due to the effects of proton accumulation in the microenvironment (in the reaction catalysed by alkaline phosphatase, protons are produced), to limitations to the free diffusion of H+ and to the possible partition effects of H+ due to polar interactions with residues or molecules of the enzyme membrane. In the experimental working conditions, the apparent optimum temperatures are centered at 40 degrees C, inactivation (thermal denaturation) occurring above this temperature. In the temperature range 10-40 degrees C, the kinetic control over the overall activity of the immobilized enzyme was observed, causing the Arrhenius profiles to be linear.

Catalytically active monoamine oxidase type A from human liver expressed in Saccharomyces cerevisiae contains covalent FAD

Monoamine oxidase type A from human liver cDNA was expressed in Saccharomyces cerevisiae. This enzyme's properties with respect to Km and Ki values for kynuramine and amphetamine, respectively, were similar to values for human placental enzyme. As expected, clorgyline inhibited the yeast enzyme at lower concentrations than deprenyl. Interestingly, the FAD cofactor was covalently attached and fluorescence properties of the enzyme bound prosthetic group indicate that it is attached to a cysteine residue, the same linkage observed in other monoamine oxidases. The yield of expressed enzyme is about 15 mg/l of culture with an A600 of 15. It is suggested that covalent flavin attachment proceeds by an autoflavination mechanism.

Isolation of rat renal NAD +-dependent 15-hydroxyprostaglandin dehydrogenase

Rat renal NAD +-dependent 15-hydroxyprostaglandin dehydrogenase was purified to apparent homogeneity in the present study. The purification included ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, Blue Sepharose CL-6B column chromatography. Sephadex G-100 column chromatography and Mono-P isoelectrofocusing column chromatography. Among the chromatographies used, Mono-P chromatofocusing column of fast protein liquid chromatography gave the most powerful resolution. The enzyme was eluted at pH 6.75 on chromatofocusing column. It indicated that the rat renal enzyme is an acidic protein. Its molecular weight in SDS-polyacrylamide gel electrophoresis was 28 Kd, and the molecular weight of the enzyme having enzyme activity detected by gel filtration column chromatography was 55 Kd. For comparing the characteristics of rat renal enzyme with that of human placental enzyme, polyclonal antibodies and a monoclonal antibody against human placental enzyme were used. The rat renal enzyme did not react with the polyclonal antibodies of human placental enzyme, however, it reacted with a monoclonal antibody of human placental enzyme. The results indicated that the antigenecity of rat renal NAD +-dependent 15-hydroxyprostaglandin dehydrogenase is different from that of human placental enzyme. The amino acid composition of rat renal enzyme was also different from that of human placental enzyme. Nine tyrosine molecules were observed in the subunit of rat renal enzyme, while no tyrosine has been reported in human placental enzyme.

Cloning and sequence analysis of the cDNA for human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.

Affinity alkylation of human placental 3β -hydroxy-5-ENE-steroid dehydrogenase and steroid 5→4-ene-isomerase by 2α-bromoacetoxyprogesterone: Evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5→4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH 2-terminal sequencing data. The affinity alkylator, 2α-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C 21 and C 19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations ( 50 1 − 20 1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity ( t 1 2 = 1.5−3.7 min ) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepi- androsterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2α-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism ( k′ 3 = 160.91/mol·s), while the alkylator inactivates isomerase by a unimolecular mechanism ( K i =0.14mM, k 3 =0.013 s −1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2α-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD + protects neither. From Dixon analysis, NADH competitively inhibits NAD + reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD + and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C 21 and C 19 isomerase activities at a second site on the same protein.