Gary L. Murdock

Isolation and sequencing of a complementary deoxyribonucleic acid clone encoding human placental 173-estradiol dehydrogenase: Identification of the putative cofactor binding site

17 beta-Estradiol dehydrogenase (EC 1.1.1.62) catalyzes the interconversion of estradiol and estrone in human term placenta. We have raised a specific polyclonal antibody to this abundant placental enzyme to study its role in late pregnancy events and its molecular biologic characteristics. In this work the 17 beta-estradiol dehydrogenase antibody was used to isolate and sequence a complementary deoxyribonucleic acid clone encoding about 98% of the amino acid sequence of the 17 beta-estradiol dehydrogenase molecule. This sequence verifies previous sequence data on the molecules steroid binding site and also localizes a putative nicotinamide adenine dinucleotide binding region similar to that of many other pyridine nucleotide-dependent dehydrogenases. Isolation of the complementary deoxyribonucleic acid for 17 beta-estradiol dehydrogenase expands our knowledge of the structure-function relationships of the enzyme and is a major step in our understanding of its biologic function in pregnancy.

Hydrogen bonding in steroidogenesis: Studies on new heterocyclic analogs of estrone that inhibit human estradiol 17β-dehydrogenase

New heterocyclic analogs of estrone are reported that inhibit estradiol 17 beta-dehydrogenase (E2-17 beta DH) from human placenta. The inhibitors are efficiently synthesized in two steps from estrone (or its 3-O-methyl ether), giving fully characterized analogs with pyrazole or isoxazole fused to the 16,17-position on the D ring. Dixon plots of enzyme kinetic data show the heterocyclic steroids are competitive inhibitors of E2-17 beta DH. Correlating molecular structures of the inhibitors with their Ki-values yields a pattern suggesting intermolecular hydrogen bonding stabilizes the [(pyrazole)inhibitor-E2-17 beta DH] complexes. A free energy difference of 2.74 Kcal/mol calculated from Ki-value differences between hydrogen bonded (4.08 microM) and non-bonded (425 microM) [inhibitor-E2-17 beta DH] complexes is in the range for intermolecular hydrogen bonding. We conclude that specific intermolecular hydrogen bonds stabilize [hydroxysteroid-enzyme] complexes, thereby making important contributions to the affinity between hydroxysteroids and steroid-specific enzymes of steroidogenesis.

A Potential Role for Aldose Reductase in Steroid Metabolism

Aldose reductase (ALR2) is a monomeric NADPH-dependent reductase distinguished from other members of the aldo-keto reductase enzyme family in its ability to catalyze the reduction of a variety of hexoses and pentoses. The role of ALR2 in enhanced polyol synthesis in diabetic and galactosemic tissues is well documented (Kinoshita and Nishimura, 1988), as is the potential therapeutic use of ALR2 inhibitors to delay or prevent the onset and progression of metabolic complications leading to cataract and retinopathy (Sarges and Oates, 1993). New insights into the structure of ALR2, together with emerging data demonstrating that many individual aldo-keto reductases may participate in divergent metabolic pathways, lead us to question whether sugars represent the only physiologically-relevant substrate of this enzyme.

Estradiol 17β-dehydrogenase : full enzymatic activity in the absence of zinc

The precise catalytic mechanism of the steroid interconverting enzyme, human placental estradiol 17β-dehydrogenase (EC 1.1.1.62, estradiol-17β:NAD+17-oxidoreductase), is not known. Two general models for the catalytic mechanism of dehydrogenase have been defined. One model requires Zn2+ metal for the catalytic event, as has been shown for horse liver alcohol dehydrogenase (EC 1.1.1.1, alcohol: NAD+ oxidoreductase). Another model has been demonstrated for the 2-hydroxy acid dehydrogenases in which histidine residues are necessary for enzyme activity, without participation of a metal ion. In order to define which mechanism might be operative for the placental enzyme, it became important to determine whether Zn2+, or another metal ion, is associated with the macromolecule. Several homogeneous enzyme preparations, having protein concentrations from 5–80 μM, were extensively dialyzed in a buffer containing EDTA. Atomic absorption analysis of each sample demonstrated that no Zn2+ was present, although the enzymatic activity was maintained. In addition, there was no significant detection of Mg2+ or Mn2+ above background levels. When the isolated enzyme was dialyzed against buffer containing added 0.01–20 μM ZnCl2, no increase in specific activity of the enzyme was seen. The data indicate that the presence of zinc is not required for the catalytic event. These results, together with our previous affinity-labeling studies, which demonstrate a histidine residue in the catalytic region of the active site, allow us to propose that the catalytic mechanism of the human placental estradiol 17β-dehydrogenase is similar to that of the 2-hydroxy acid dehydrogenases.

Stereospecificity of hydrogen transfer by bovine testicular 20α-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.

Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17β dehydrogenase

We have previously shown that human placental estradiol-17 beta dehydrogenase (EC 1.1.1.62; 17 beta-EDH) catalyzes the conversion of estradiol-17 beta to estrone and stereospecifically reduces NAD+ to [4-pro-S]NADH, [( 4-B]NADH). Subsequently, this enzyme was found to reduce the ketone function at C-20 of progesterone, and evidence indicates that both activities reside at the same active site. This study was done to further elucidate spatial arrangements of cofactor and the 21-carbon substrate as they bind at the active site. The cofactor, [4B-3H]NADPH, was generated with homogeneous 17 beta-EDH from term human placenta, utilizing [17 alpha-3H]estradiol-17 beta and NADP+. The resulting [4B-3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 microM) with a large molar excess of progesterone (150 microM) as substrate in the presence of the enzyme. Following incubation, the steroid reactants and products were extracted, separated by high-performance liquid chromatography and quantitated as to mass and tritium content. Oxidized and reduced cofactor were separated by ion-exchange chromatography and similarly quantitated. In all incubations, equimolar amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OHP) and NADP+ were obtained. Radioactivity was stoichiometrically transferred from [4B-3H]NADPH to the steroid product [( 3H]20 alpha-OHP). These results further substantiate a single active site for both 17 beta- and 20 alpha-dehydrogenation enzyme activities. In addition, the enzyme is B-side specific, catalyzing the transfer of the 4B-hydrogen from the dihydronicotinamide moiety of the cofactor, for both C-18 and C-21 steroid substrates. Since the 20 alpha-dehydrogenation by other enzyme sources has always been demonstrated to be an A-side specific reaction, this observation represents an important exception to the Alworth-Bentley rules of enzyme stereospecificity.