The use of fluorescein 5'-isothiocyanate for studies of structural and molecular mechanisms of soybean lipoxygenase

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) plays a crucial role in the last step of the synthesis of estrogens. A detailed kinetic study demonstrated that the enzyme shows about 240 fold higher specificity towards estrone reduction than estradiol oxidation at physiological pH using tri-phosphate cofactors. The kcat/Km values are 96 +/- 10 and 0.4 +/- 0.1 s-1 (microM)-1 respectively for the above two reactions. However, it has been shown that this difference is closely linked to the use of NADPH and NADP cofactors. A binding study using equilibrium dialysis indicated similar KD (equilibrium dissociation constant) of 11 +/- 1 and 4.7 +/- 0.9 microM for estrone and estradiol, respectively. The binding affinity of 17beta-HSD1 to estrone was significantly increased with a KD of 1.6 +/- 0.2 microM in the presence of NADP, the latter used as an analogue of the NADPH. The results of binding studies agree with the steady-state kinetics, which showed that the Km of estrone is 12-fold lower when using NADPH as a cofactor than when using NADH. These results strongly suggest that the cofactor plays a crucial role in the stimulation of the specificity for estrogen reduction.     

Monte Carlo-minimized energy profile of estradiol in the ligand-binding tunnel of 17 beta-hydroxysteroid dehydrogenase: atomic mechanisms of steroid recognition

17 beta-Estradiol (E2) is a potent stimulator of certain forms of breast cancer. The final step of E2 biosynthesis is catalyzed by the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), which is an important target for anti-cancer drugs. X-ray crystallography indicated that the binding site for the steroids has a tunnel-like shape. We have used a Monte Carlo-Minimization (MCM) protocol to explore possibilities of interactions of E2 with the binding site tunnel of 17 beta-HSD1. The enzyme was represented by flexible residues having at least one atom within 6 A from either E2 or NADP (as seen in a crystal ternary complex) and by rigid residues having at least one atom within 10 A from E2 or NADP. Special constraints were used to pull the substrate 10 A along the tunnel with 1 A step; the complex was MCM-optimized at each position of the steroid. The optimal binding mode of E2 in 17 beta-HSD agrees with the crystallographic data; however, wide and flat minima of the MCM profile suggest alternative modes of the steroid binding. The advance of the steroid along the tunnel is accompanied by essential conformational rearrangements of the enzyme side chains, noticeable rotation of the substrate along its longitudinal axis, and certain conformational deformations of the substrate. The contributions of the enzyme residues and of the steroid atoms to the intermolecular energy were estimated.     

Chemical synthesis of 16beta-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17beta-hydroxysteroid dehydrogenase and binding affinity on steroid receptors

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.     

Functional genome analysis indicates loss of 17beta-hydroxysteroid dehydrogenase type 2 enzyme in the zebrafish

Among the family of 17beta-hydroxysteroid dehydrogenases, the type 2 (17beta-HSD 2) is the main enzyme responsible for inactivation of estrogens and androgens, catalyzing the oxidation of the C17 hydroxyl group. 17beta-HSD 2 has been studied only in mammals, its occurrence and function in other vertebrates hardly known. We investigated the presence of homologs in non-mammalian species and found sequences of 17beta-HSD 2 and its closest homolog 11beta-HSD 2 in zebrafish (Danio rerio), Takifugu rubripes, Tetraodon nigroviridis, Xenopus tropicalis and chicken databases. Furthermore, we cloned zebrafish 17beta-HSD 2 from ovarian tissue and found high expression also in the testis of adult fish and throughout embryogenesis. The enzyme, though, is inactive likely due to a non-sense N-terminal region including a dysfunctional cofactor binding motif. Replacement of the affected part by the corresponding human 17beta-HSD 2 sequence fully restored enzymatic activity. Comparison of all retrieved 17beta-HSD 2 sequences indicates that this functional loss may have occurred only in zebrafish, where steroid inactivation at position C17 seems to pursue without the protein studied. The closely related 11beta-HSD 2 is unlikely to substitute for 17beta-HSD 2 since in our hands it did not catalyze the respective oxidation of testosterone or estradiol.     

Calorimetric studies of ligand binding in R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.     

Inhibitors of type 1 17beta-hydroxysteroid dehydrogenase with reduced estrogenic activity: modifications of the positions 3 and 6 of estradiol

Breast cancer is the second most frequent cancer affecting women. Among all endocrine therapies for the treatment of breast cancer, inhibition of estrogen biosynthesis is becoming an interesting complementary approach to the use of antiestrogens. The enzyme type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD) plays a critical role in the biosynthesis of estradiol catalyzing preferentially the reduction of estrone into estradiol, the most active estrogen. Consequently, this enzyme is an interesting biological target for designing drugs for the treatment of estrogen-sensitive diseases such as breast cancer. Our group has reported the synthesis and the biological evaluation of N-methyl, N-butyl 6beta-(thiaheptamamide)estradiol as a potent reversible inhibitor of type 1 17beta-HSD. Unfortunately, this inhibitor has shown an estrogen effect, thus reducing its possible therapeutic interest. Herein three strategies to modify the biological profile (estrogenicity and inhibitory potency) of the initial lead compound were reported. In a first approach, the thioether bond was replaced with a more stable ether bond. Secondly, the hydroxyl group at position 3, which is responsible for a tight binding with the estrogen receptor, was removed. Finally, the amide group of the side-chain was changed to a methyl group. Moreover, the relationship between the inhibitory potency and the configuration of the side-chain at position 6 was investigated. The present study confirmed that the 6beta-configuration of the side chain led to a much better inhibition than the 6alpha-configuration. The replacement of the 3-OH by a hydrogen atom as well as that of the amide group by a methyl was clearly unfavorable for the inhibition of type 1 17beta-HSD. Changing the thioether for an ether bond decreased by 10-fold the estrogenic profile of the lead compound while the inhibitory potency on type 1 17beta-HSD was only decreased by 5-fold. This study contributes to the knowledge required for the development of compounds with the desired profile, that is, a potent inhibitor of type 1 17beta-HSD without estrogen-like effects.     

Amino acid substitution of arginine 80 in 17beta-hydroxysteroid dehydrogenase type 3 and its effect on NADPH cofactor binding and oxidation/reduction kinetics

17beta-Hydroxysteroid dehydrogenase type 3 (17beta-HSD-3) is a member of the short-chain dehydrogenase/reductase (SDR) family and is essential for the reductive conversion of inactive C(19)-steroid, androstenedione, to the biologically active androgen, testosterone, which plays a central role in the development of the male phenotype. Mutations that inactivate this enzyme give rise to a rare form of male pseudohermaphroditism, referred to as 17beta-HSD-3 deficiency. One such mutation is the replacement of arginine at position 80 with glutamine, compromising enzyme activity by increasing the cofactor binding constant 60-fold. In the absence of a 17beta-HSD-3 crystal structure, we have grafted its amino acid sequence for the NADPH binding site on the X-ray crystal structures of glutathione reductase (Protein Data Bank code 1gra) and 17beta-HSD type 1 (Protein Data Bank codes 1fdv and 1fdu) where we find the trunk of the arginine 80 side chain forms part of the hydrophobic pocket for the purine ring of adenosine while its guanidinium moiety interacts with the 2'-phosphate to both stabilize cofactor binding and neutralize its intrinsic negative charge through two hydrogen bonds. To qualitatively assess the role arginine 80 plays in both selecting and stabilizing NADPH binding, it was replaced with each amino acid and the mutant enzymes subjected to enzymatic analysis. There are only seven enzymes exhibiting any measurable enzymatic activity with arginine approximately lysine>leucine>glutamine>methionine>tyrosine>isoleucine. With an aspartic acid at position 58 in 17beta-HSD-3 occupying the equivalent space in the cofactor binding pocket as arginine 224 in glutathione reductase or serine 12 in 17beta-HSD-1, there was an expectation that some of the mutants might use NADH as a cofactor. In no case was NADH found to substitute for NADPH.     

Crystal structures of the multispecific 17beta-hydroxysteroid dehydrogenase type 5: critical androgen regulation in human peripheral tissues

Human type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD5;AKR1C3) plays a major role in the metabolism of androgens in peripheral tissues. In prostate basal cells, this enzyme is involved in the transformation of dehydroepiandrosterone into dihydrotestosterone, the most potent androgen. It is thus a potential target for prostate cancer therapy because it is understood that the testosterone formation by this enzyme is an important factor, particularly in patients who have undergone surgical or medical castration. Here we report the first structure of a human type 5 17beta-HSD in two ternary complexes, in which we found that the androstenedione molecule has a different binding position from that of testosterone. The two testosterone-binding orientations in the substrate-binding site demonstrate the structural basis of the alternative binding and multispecificity of the enzyme. Phe306 and Trp227 are the key residues involved in ligand recognition as well as product release. A safety belt in the cofactor-binding site enhances nicotinamide adenine dinucleotide phosphate binding and accounts for its high affinity as demonstrated by kinetic studies. These structures have provided a dynamic view of the enzyme reaction converting androstenedione to testosterone as well as valuable information for the development of potent enzyme inhibitors.     

Collective motions in glucosamine-6-phosphate synthase: influence of ligand binding and role in ammonia channelling and opening of the fructose-6-phosphate binding site

The large protein motions of the bacterial enzyme glucosamine-6-phosphate synthase have been addressed using full atom normal modes analysis for the empty, the glucose-6-phosphate and the glucose-6-phosphate + glutamate bound proteins. The approach that was used involving energy minimizations along the normal modes coordinates identified functional motions of the protein, some of which were characterized earlier by X-ray diffraction studies. This method made it possible for the first time to highlight significant energy differences according to whether none, only one or both of the active sites of the protein were occupied. Our data favoured a specific motion of the glutamine binding domain following the fixation of fructose-6-phosphate and suggested a rigidified structure with both sites occupied. Here, we show that most of the collective large amplitude motions of glucosamine-6-phosphate synthase that are modulated by ligand binding are crucial for the enzyme catalytic cycle, as they strongly modify the geometry of both the ammonia channel and the C-tail, demonstrating their role in ammonia transfer and ligand binding.     

Ligand binding to proteins: the binding landscape model.

Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand-induced denaturation, ANS and heme-like binding, and "lock-and-key" and "induced-fit" specific binding behaviors characterized by Michaelis-Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis-Menten isotherms. Even low-population binding to denatured states can cause changes in global stability, hydrogen-exchange rates, and thermal B-factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass-action models.     

Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa

The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.     

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force‐field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen‐bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints.     

Role of 17 beta-hydroxysteroid dehydrogenase in the modulation of nuclear estradiol receptor binding by progesterone in the rat anterior pituitary gland and the uterus

Progesterone has been shown to decrease occupied pituitary and uterine nuclear estradiol receptor (E2R) binding in mature and immature estrogen-primed rats. Progesterone has also been shown to stimulate pituitary but not uterine 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the rat. The conversion of estradiol to its less active metabolite estrone by 17 beta-HSD and activation of phosphatase are among mechanisms considered to be involved in the reduction of E2R. To determine if 17 beta-HSD stimulation was a mechanism by which progesterone induced nuclear E2R decrease, the synthetic estrogen ethinylestradiol, which is not oxidized by 17 beta-HSD, was used instead of estradiol to prime adult ovariectomized rats. When ethinylestradiol-primed rats received 0.8, 2.0 or 4.0 mg/kg body wt of progesterone 2 h before sacrifice, the total and occupied nuclear E2R accumulation in the anterior pituitary by a subsequent ethinylestradiol injection 1 h later did not show any decrease. This response was different from that observed previously in estradiol-primed animals in which progesterone showed a multiphasic decrease of occupied form of nuclear E2R. However, in the uterus of ethinylestradiol-primed rats, a partial decrease of total and occupied nuclear E2R accumulation was observed in the presence of the three doses of progesterone used. The decrease of uterine nuclear E2R with the three progesterone doses was different from the dose-dependent effect of progesterone observed in the uterus of estradiol-primed rats. Affinity constants of the interaction between [3H]estradiol and the nuclear E2R were similar among groups treated with ethinylestradiol, estradiol and progesterone. These results demonstrate the involvement of 17 beta-HSD in the reduction of anterior pituitary gland E2R by progesterone in the estradiol-treated animals. Furthermore, the mechanism of decrease of E2R by progesterone in the uterus appears to be different from the pituitary gland.     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.     

The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism.

The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.     

3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties.

The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.     

Dehydroepiandrosterone and dihydrotestosterone recognition by human estrogenic 17beta-hydroxysteroid dehydrogenase. C-18/c-19 steroid discrimination and enzyme-induced strain

Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. The K(m) of estrone is more than 1000-fold lower than that of DHEA and the K(m) of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17beta-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 --> Val variant showed a significantly decreased K(m) for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a beta-oriented form, which approaches the transition-state conformation for DHEA reduction.     

Stimulation of 17 beta-hydroxy steroid dehydrogenase in the rat anterior pituitary gland by progesterone

Anterior pituitary gland and hypothalamic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was measured in the immature castrated estradiol primed rat to determine if differences in enzyme activity could explain the progesterone induced reduction of bound estradiol nuclear receptors of the anterior pituitary gland but not the hypothalamus. Higher levels of 17 beta-HSD activity were found in the anterior pituitary gland as compared to the hypothalamus. The enzyme activity in the anterior pituitary gland was stimulated by progesterone administered either in combination with estradiol for 4 days or as a single injection following 4 days of estradiol priming. No progesterone effects were found on hypothalamic 17 beta-HSD. Under the experimental conditions used, progesterone administration did not alter uterine 17 beta-HSD. An increase in anterior pituitary gland and uterine 17 beta-HSD was also induced by estrogen administration.     

Dynamics of carbon monoxide binding to cystathionine beta-synthase

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.     

Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.