首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E1), estradiol (E2), 16-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-3H]testosterone (1.24 × 103 dpm 3H/ng, 35–150 nM) or [1β-3H,4-14C]androstenedione (3.05 × 103 dpm 3H/ng, 3H/14C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E1, E2, 16-OHE1, and E3 for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the Km of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The Ki of E1, E2, 16-OHE1, and E3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the Km of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.  相似文献   

2.
Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3-O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3-O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3-O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.  相似文献   

3.
Currently there is much interest in the role that growth factors may play in the development of human breast tumours. We have shown previously that growth factors secreted by breast tumours may influence the activity of oestradiol hydroxysteroid dehydrogenase, the enzyme which catalyses the interconversion of oestrone (E1) and oestradiol. As the formation of E1 from its sulphate (E1S) by oestrone sulphatase may be quantitatively more important than production from androstenedione via aromatase, we have studied the effect of insulin-like growth factor-1 (IGF-I) and basic fibroblast growth factor (bFGF) on oestrone sulphatase activity in the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231 breast cancer cell lines. In both these cell types, bFGF (1–200 ng/ml) and IGF-I (25–200 ng/ml) significantly stimulated oestrone sulphatase activity in a dose-dependent manner (by 8–60%) after 48 h. Additionally, cycloheximide significantly inhibited (by 90–120%) this stimulation of oestrone sulphatase activity by the two growth factors in both MCF-7 and MDA-MB-231 cells. Basal oestrone sulphatase activity was higher in the oestrogen receptor, ER - ve MDA-MB-231 cells than in the ER + ve MCF-7 breast cancer cells. We conclude that these growth factors, believed to be secreted by breast tumours, may induce enzymes of oestrogen synthesis and hence increase local production of oestrogens.  相似文献   

4.
The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [3H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The Km value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the Vmax value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (Ki = 0.9 ± 0.4 μM; N = 4), followed by gestodene (Ki = 5.6 ± 0.6 μM) and cyclosporine (Ki = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (Ki = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the Vmax value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (Ki = 17.8 ± 3.3 μM) and gestodene (Ki = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.  相似文献   

5.
Polyphosphates of different chain lengths (P3, P4, P15, P35), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p4A and P4 (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap4A was not inhibitory. P4 and P15 were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P4 and P15 behaved as competitive inhibitors, with Ki values of 0.5 μM and 0.2 μM, respectively. In addition, P4 (at 1 μM) and P15 (at 0.3 μM) changed the Hill coefficient (nH) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P4 and P15 decreased V and modified only slightly the Km values of the enzyme towards tRNA.  相似文献   

6.
To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (Ki < 1 × 10−9 M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (Ki = 6 × 10−8 M) > 5β-androstanedione > 5β-dihydrotestosterone (Ki = 3.5 × 10−7 M) > 5-androstane-3, 17β-diol (Ki = 5 × 10−6 M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10−4 M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.  相似文献   

7.
3β-hydroxysteroid dehydrogenase 5-ene isomerase (3βHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3βHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (Km=0.17±0.03 and 1.02 μM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected Vmax while Km was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. Ki was calculated from replot of Dixon's slope for each substrate concentration. Ki from the intercept and the slope of this replot were similar (0.276±0.01 and 0.263±0.02 μM) and higher than the Km for DHEA. The Km and Ki values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the Vmax while Km values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.  相似文献   

8.
Steroid sulphatases regulate the formation of oestrogenic steroids which can support the growth of endocrine-dependent breast tumours. The development of potent steroid sulphatase inhibitors could therefore have considerable therapeutic potential. Several such inhibitors have now been developed of which the most potent to date is oestrone-3-O-sulphamate (EMATE). Unexpectedly, this inhibitor proved to be a potent oestrogen. In an attempt to reduce the oestrogenicity, whilst retaining the potent sulphatase inhibitory properties associated with this type of molecule, a number of A-ring modified derivatives were designed and synthesized. A-ring modified compounds included the 2-methoxy, 2/4-nitro, 2/4-n-propyl and 2/4-allyl EMATE analogues. The ability of these derivatives to inhibit oestrone sulphatase activity was examined using placental microsomes. The allyl-substituted EMATE derivatives were more potent inhibitors than the propyl analogues but were all considerably less potent than EMATE. In contrast, the 2-methoxy and 2/4-nitro analogues were potent sulphatase inhibitors with 4-nitro EMATE being 5 times more active than EMATE. The 4-nitro, 2-methoxy, 4-n-propyl and 4-allyl derivatives were also tested in vivo for their oestrogenicity and ability to inhibit sulphatase activity. While both 4-nitro and 2-methoxy EMATE were potent inhibitors in vivo, 2-methoxy EMATE had no stimulatory effect on uterine growth in ovariectomized rats. The identification of a potent steroid sulphatase inhibitor lacking any oestrogenicity, such as 2-methoxy EMATE, should be of considerable value in evaluating the potential of steroid sulphatase inhibition for breast cancer therapy.  相似文献   

9.
The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an -proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E2 site), but is in a different location in two other family members (the E1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E2 to the E1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (Km = 0.6 and 2.0 μM, and Vmax = 2.8 and 0.3 s−1 for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis—abstraction of the -HR from the substrate followed by transfer of the β-HR to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E1 and E2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform γ-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the -HR but is far removed from the γ-Hs. These findings provide further support for a one base mechanism of - and γ-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.  相似文献   

10.
Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (Km = 17.9 μM) and placental microsomes (Km = 16.7 μM). The 3β-HSD activity (Vmax = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (Vmax = 9.1 nmol/min/mg). The Km values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (Km = 14.7 μM) and placental microsomes (Km = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (Vmax = 25.7 nmol/min/mg) relative to placenta (Vmax = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.  相似文献   

11.
An overview of the application of kinetic methods to the delineation of 17β-hydroxysteroid dehydrogenase (17β-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17β-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17β-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17β-HSD type 1 is a cytosolic enzyme highly specific for C18 steroids such as 17β-estradiol (E2) and estrone (E1). 17β-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E2 and E1. Useful parameters for the detection of multiple forms of 17β-HSD appear to be the E2/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17β-HSD type 2. The 17β-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17β-HSD which differ from 17β-HSD type 1 and type 2 in their kinetic properties.  相似文献   

12.
13.
Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the KD and Bmax of the BK receptor for binding (control: KD = 1.7 ± 0.2 nM; Bmax = 47.3 ± 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.  相似文献   

14.
Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed Mr of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg−1 (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min−1·mmg−1 and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min−1·mg−1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 μM and Vmax of 8.9 nmol·min−1·mg−1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed Ki of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.  相似文献   

15.
Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (Ki 53 μM) and ciprofibrate (Ki 1000 μM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (Km 6 ± 2.5 μM, (Vmax 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean Ki 1.7 μM, n = 5) and R-ibuprofen (mean Ki 10.8 μM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (Vmax/Km 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the Km and Ki for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.  相似文献   

16.
The use of specific and non-specific antisera for estradiol-17β (E217β) were compared in the radioimmunoassay of the steroid. The effects of various “blank” mateirials on the standard curve and on the accuracy of recovery of E217β added to plasma before and after chromatography on LH-20 Sephadex were examined. It was concluded that the use of the specific antiserum (anti-6-oxoE217β -6-(O-carboxymethyl)oxime-bovine serum albumin(antiE217β-6-BSA) was an improvement on the non-specific serum anti-E217β-17-hemisuccinyl-bovine serum albumin (antiE2 17β-17-BSA) following chromatography of extracts. However, although a precise result could be obtained with the anti-E217β-6-BSA without the Chromatographic step, recovery of E217β added to plasma was only possible if the step was included.

The cross-reactivity of estrone (E1)with E217β using anti-E217β-17-BSA as defined by Abraham (J. Clin. Endocr. , 866 (1969) was examined under conditions of constant and of changing E1:E217β ratio.  相似文献   


17.
N,N-Dialkylated derivatives of the steroid sulphatase inhibitor, oestrone 3-O-sulphamate (EMATE) are weak reversible inhibitors of the enzyme. N-Acetylated-EMATE (8), but not the benzoyl derivative, inhibits the enzyme irreversibly, albeit less potently than EMATE and will allow hitherto difficult radiolabelling on the sulphamate group to facilitate investigation of the enzyme inactivation mechanism.  相似文献   

18.
A series of dihydroxamic acid ligands of the formula [RN(OH)C(O)]2(CH2)n, (n = 2, 4, 6, 7, 8; R = CH3, H) has been studied in 2.0 M aqueous sodium perchlorate at 25.0 °C. These ligands may be considered as synthetic analogs to the siderophore rhodotorulic acid. Acid dissociation constants (pKa) have been determined for the ligands and for N-methylacetohydroxamic acid (NMHA). The pKa1 and pKa2 values are: n = 2, R = CH3 (8.72, 9.37); N = 4, R = CH3 (8.79, 9.37); N = 6, R = CH3; N = 7, R = CH3 (8.95, 9.47); N = 8, R = CH3 (8.93, 9.45); N = 8, R = H (9.05, 9.58). Equilibrium constants for the hydrolysis of coordinated water (log K) have been estimated for the 1:1 feeric complexes of the ligands n = 2, 4, 8; R = CH3. The N = 8 ligand forms a monomeric complex with Fe(III) while the n = 2 and 4 ligands form dimeric complexes. For hydrolysis of the n = 8 monomeric complex, log K1 = −6.36 and log K2 = −9.84. Analysis of the spectrophotometric data for the dimeric complexes indicates deprotonation of all four coordinated waters. The successive hydrolysis constants, log K1–4, for the dimeric complexes are as follows: n = 2 (−6.37, −5.77, −10.73, −11.8); n = 4 (−5.54, −5.07, −11.57, −10.17). The log K2 values for the dimers are unexpectedly high, higher in fact than log K1, inconsistent with the formation of simple ternary hydroxo complexes. A scheme is proposed for the hydrolysis of the ferric dihydroxamate dimers, which includes the possible formation of μ-hydroxo and μ-oxo bridges.  相似文献   

19.
The photoreactive nucleotide 3'-O-(4-benzoyl)benzoyl ADP (BzADP) is not a substrate for photophosphorylation but is a strong competitive inhibitor (Ki 2-25μM) with respect to ADP and ATP in photophosphorylation or ATP hydrolysis and Pi-ATP exchange reactions, respectively. The analog binds tightly to the membrane-bound CF1, competes with the right binding of ADP, and prevents the inactivation of the enzyme by tight binding of ADP. Upon irradiation with long wavelength ultraviolet light, the tightly bound BzADP becomes covalently attached to both the - and β-subunits of the enzyme.  相似文献   

20.
Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment of steroid-dependent cancers. Two steroidal and two coumarin- and chromenone-based boronic acids were synthesized and examined as inhibitors of purified STS. The boronic acid analog of estrone sulfate bearing a boronic acid moiety at the 3-position in place of the sulfate group was a good competitive STS inhibitor with a Ki of 2.8 μM at pH 7.0 and 6.8 μM at pH 8.8. The inhibition was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. An estradiol derivative bearing a boronic acid group at the 3-position and a benzyl group at the 17-position was a potent reversible, non-competitive STS inhibitor with a Ki of 250 nM. However, its 3-OH analog, a known STS inhibitor, exhibited an almost identical affinity for STS and also bound in a non-competitive manner. It is suggested that these compounds prefer to bind in a hydrophobic tunnel close to the entrance to the active site. The coumarin and chromenone boronic acids were modest inhibitors of STS with IC50s of 86 and 171 μM, respectively. Surprisingly, replacing the boronic acid group of the chromenone derivative with an OH group yielded a good reversible, mixed type inhibitor with a Ki of 4.6 μM. Overall, these results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号