Binding of the chymotrypsin substrate,tryptophan methyl ester,by rat α-fetoprotein

We studied how tryptophan methyl ester and related compounds inhibit binding of estrone to rat α-fetoprotein and find that: (a) like chymotrypsin, α-fetoprotein binds tryptophan esters with higher affinity than tryptophan or its amides; (b) the affinity of α-fetoprotein for tryptophan methyl ester is 3.7 · 10^?4 M, which is close to the affinity of chymotrypsin (10^?4 M); (c) α-fetoprotein binding of tryptophan methyl ester is stereoselective and pH dependent. All of these observations suggest that there is a specific interaction between α-fetoprotein and the chymotrypsin substrate, tryptophan methyl ester, and that rat α-fetoprotein contains a site with some structural similarities to the catalytic site in chymotrypsin. Since we also find that tryptophan methyl ester is a competitive inhibitor of estrone binding to α-fetoprotein, it is possible that the protease substrate binding site on α-fetoprotein is spatially close to the estrone binding site.     

Diethylpyrocarbonate inhibition of estrogen binding to rat alpha-fetoprotein: evidence that one or more histidine residues regulate estrogen binding

Rat alpha-fetoprotein contains a site that both binds serine enzyme inhibitors and substrates and regulates estrogen binding. We report that mM concentrations of the histidine selective reagent, diethylpyrocarbonate, inhibit estrogen binding to rat alpha-fetoprotein and that this inhibition is reversed by hydroxylamine. We suggest that rat alpha-fetoprotein contains one or more histidine residues that regulate estrogen binding. We also find that either estrone or the chymotrypsin substrate, acetyl-tryptophan methyl ester, protects rat alpha-fetoprotein from diethyl-pyrocarbonate-mediated inhibition of estrogen binding. We infer that the protease substrate and estrogen binding sites contain histidine residue(s) essential for estrogen binding by alpha-fetoprotein.     

Interaction of serine protease inhibitors and substrates with human uterine estrogen receptor

The human uterine estrogen receptor has a site which regulates estrogen binding and which structurally resembles the substrate binding site of chymotrypsin. The hormone binding capacity and the affinity of the receptor is decreased in the presence of 4 mM serine protease inhibitors tosyl-lysine chloromethyl ketone and diisopropylfluorophosphate and the protease substrates tryptophan methyl ester and toluene sulphonyl-arginine methyl ester. The protease inhibitors tosylamide-phenylethyl-chloromethyl ketone and phenyl methyl sulphonyl fluoride caused an increase in the binding capacity whereas the affinity was decreased.     

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.     

Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents

Initial rates of peptide-bond synthesis catalyzed by poly(ethylene glycol)-modified chymotrypsin in benzene were determined using high-performance liquid chromatography. Enzymatic synthesis of N-benzoyl-L-tyrosyl-L-phenylalanine amide from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine amide was found to obey Michaelis-Menten kinetics an to be consistent with a ping-pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N-benzoyl-L-tyrosine ethyl ester and N-alpha-benzoyl-L-lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10-fold smaller than that obtained with poly(ethylene glycol)-modified trypsin in the synthesis of N-alpha-benzoyl-L-lysyl-L-phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme-binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.     

A surprising increase in the affinity of estrogen for rat alpha-fetoprotein after it is treated with N-ethylmaleimide

Incubation of rat alpha-fetoprotein (AFP) with mM concentrations of N-ethylmaleimide (MalNEt) at 22 degrees, pH7.8 for several hours yields a form of AFP with an equilibrium dissociation constant of 0.1 nM for estrone. This affinity for estrone is about 10 fold higher than that for untreated rat AFP. MalNEt-treated AFP still binds inhibitors and substrates of serine proteases suggesting that the binding of these compounds to AFP does not involve an interaction with a thiol group. MalNEt-treated AFP may be useful for studying the mechanism of estrogen action in vitro and in vivo, estrogen-protein interactions, and the functioning of AFP.     

Gossypol inhibits estrogen binding to rat alpha-fetoprotein

We find that gossypol, a male anti-fertility compound, is a reversible competitive inhibitor of estrogen binding to rat alpha-fetoprotein (AFP). The Kd of gossypol for rat AFP is 1.75 microM, which is similar to gossypol's affinity for lactate dehydrogenase isozyme X, the putative site where gossypol exerts its anti-fertility effects. Reacting sodium cyanoborohydride with gossypol reduces its affinity for AFP, showing that intact aldehyde groups on gossypol are important for binding to rat AFP and indicating that gossypol is specifically inter-acting with a nucleophilic site on AFP that influences estrogen binding.     

Transition state affinity jump chromatography. A double selection method for isolating catalytically active enzymes and other molecules

A double selection method for isolating active enzyme molecules, using substrate analog affinity chromatography and elution with transition state analogs, is described. To demonstrate the principle, a mixture containing native chymotrypsin and [3H]deoxychymotrypsin, in which the active site serine had been converted to [3H]alanine, was applied to a column containing immobilized D-tryptophan methyl ester. Both forms of chymotrypsin were retained. Catalytically active enzyme was selectively desorbed with the peptide aldehyde chymostatin, leaving catalytically inactive deoxychymotrypsin bound to the substrate analog affinity column. This affinity technique may afford a simple and general method for separating enzymes and other catalysts according to their molecular turnover numbers.     

Phytoestrogens: new ligands for rat and human alpha-fetoprotein.

The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat alpha-fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA greater than equol greater than enterolactone greater than enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants (Kd) for phytoestrogens binding to AFP were: Kd NDGA = 5 +/- 1.2.10(-7) M, Kd equol = 6.7 +/- 0.8.10(-6) M, Kd enterolactone = 1.7 +/- 0.4.10(-5) M and Kd enterodiol = 2.2 +/- 0.6.10(-5) M. The Kd for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of esterone binding sites remained unchanged. This, plus the results of double-reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased Kd), but high concentrations induced conformational changes in rat AFP, as both Kd and the number of binding sites (n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development.     

Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible substrates

A series of arylalkanoate esters and alpha-acetamidoarylalkanoate esters were tested as substrates for alpha-chymotrypsin and subtilisin BPN'. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of alpha-chymotrypsin is roughly 1.1nm (11A) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the alpha-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.     

Separation and purification of enzymes by continuous pH-parametric pumping

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. epsilon-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes.     

Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.     

A quinonoid is an intermediate of oxidative deamination reaction catalyzed by Dopa decarboxylase

The reactions of Dopa decarboxylase (DDC) with l- and d-enantiomers of tryptophan methyl ester are described. Although both the enantiomers bind to the active site of the enzyme with similar affinity, their binding modes are different. l-enantiomer binds in an unproductive mode, while d-enantiomer acts as an oxidative deamination substrate. For the first time a quinonoid has been detected as intermediate of this reaction. By using rapid-scanning stopped-flow kinetic technique rate constants for formation and decay of this species have been determined. All these data, besides validating the functional DDC active site model, represent an important step toward the elucidation of the catalytic pathway of oxidative deamination.     

Preparation of 14,15-secoestra-1,3,5(10)-trien-15-ynes, inhibitors of estradiol dehydrogenase

The conversion of estrone to 14,15-secoestratrien-15-ynes, inactivators of estradiol dehydrogenase from human term placenta, is described. The optically pure precursor 7-acetoxy-octahydro-2-phenanthrenecarboxylic acid methyl ester is prepared from estrone in five steps and 40% yield. The unsubstituted propargylic secoestratriene diol, a mechanism-based inactivator of estradiol dehydrogenase, and the corresponding acetylenic ketone, an affinity label inactivator of the same enzyme, arise from the phenanthrene ester in three and four steps. The propargylic secoestratriene diol also competes with [3H]estradiol for binding to calf uterus estrogen receptor and possesses weak uterotrophic activity.     

Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible and `locked'' substrates

Subtilisin BPN' hydrolysed N-acetyl-l-3-(2-naphthyl)-alanine methyl ester, N-acetyl-l-leucine methyl ester and N-acetyl-l-valine methyl ester, faster than alpha-chymotrypsin. Of eight ;locked' substrates tested, only methyl 5,6-benzindan-2-carboxylate was hydrolysed faster by subtilisin, whereas the other esters were better substrates for chymotrypsin. Compared with the values for chymotrypsin, the stereospecific ratios during the hydrolysis of the optically active locked substrates by subtilisin were decreased by one and two orders of magnitude for bi- and tri-cyclic substrates respectively. The polar groups adjacent to the alpha-carbon atom of locked substrates did not contribute significantly to the reactivity of the more active optical isomers, but had a detrimental effect on the less active antipodes during hydrolysis by both the enzymes. These studies show that the binding site of subtilisin BPN' is longer and broader than that of alpha-chymotrypsin.     

Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: hydrolyses of thiono and oxygen esters

X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K.H., & Swen, H.M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.     

Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

Binding of oxytocin to uterine cells in vitro. Occurrence of several binding site populations and reidentification of oxytocin receptors

Myometrial and endometrial cells of sheep, rat, and calf in monolayer cell culture display at least three populations of binding sites for oxytocin, with dissociation constants (Kd) of approximately 5 X 10(-9), 4 X 10(-7), and greater than 10(-5) mol/liter, respectively. Binding of the tritium-labeled oxytocin (concentration range, 10(-11) to 5 X 10(-4) M) to the first two sites is displaceable by cold oxytocin. The ratio of binding capacities of the high to medium affinity site appears to average 1:18. Dissociation rate constants for these sites (22 degrees C) are roughly 10(-4) and 2 X 10(-3) s-1, respectively. The capacity of the low affinity site varies in individual cell preparations and is between 5 and 66 times that of the medium affinity site. The low affinity binding sites may not be fully saturable and may follow a nonasymptotic binding isotherm. Logarithms of Kd and binding capacity for individual binding sites are linearly correlated. The coexistence of the three sites was also proven by cluster analysis based on similarities between Kd, binding capacity, and Hill coefficient. Only minor systematic species and cell type differences occur in these properties. The value of Kd for the oxytocin receptor in rat myometrium, derived recently from a stepwise irreversible inhibition of uterotonic response to oxytocin, is close to 2.5 X 10(-7) mol/liter. Additional pharmacological data (pA2 values of structural analogues of oxytocin acting as competitive inhibitors) also reveal a Kd value of 3 X 10(-7). It is, therefore, concluded that the receptors for oxytocin in rat myometrium are identical with the medium affinity site.     

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.     

Interaction between rat alpha 1 fetoprotein and fluorescent derivatives of estrone in relation to the position and type of the fluorescent label

A series of derivatives of estrone with fluorescent dyes (dansyl or coumarin) coupled at different positions on the steroid molecule, have been synthesized. These derivatives were tested for their quantum yields and their binding properties were determined with respect to rat alpha 1 fetoprotein. Derivatives at C-3 (estrone-3-hemisuccinate-dansyl-cadaverine and estrone-3-dansyl) compete with estrone for binding to the fetal protein; however derivates of estrone at C-6 (estrone-6-carboxymethyloxime-dansyl-cadaverine) and at C-17 (estrone-17-carboxymethyloxime-coumarine, estrone-17-carboxymethyloxime-dansyl-cadaverine and estrone-17-dansyl-hydrazine) compete poorly or not at all. The association constant of the radioactive derivative estrone-3-dansyl [3H] with rat alpha 1 fetoprotein was measured directly: the same number of high affinity binding sites (0.6) as that for estrone was found with an apparent association constant of 3.7 X 10(6) M-1. In addition to the high affinity binding sites, a low affinity class of binding sites was found which corresponds to the binding of the dansyl fraction of the fluorescent steroid derivative.