Inhibition of carboxypeptidase A by ketones and alcohols that are isosteric with peptide substrates

The Ki's of three peptide ketone and three peptide alcohol inhibitors of carboxypeptidase A are compared with Ki's of their respective isosteric peptide substrates, N alpha-benzoyl-L-phenylalanine, N alpha-benzoylglycyl-L-phenylalanine, and N alpha-carbobenzoxyglycylglycyl-L-phenylalanine. For the isosteric ketone analogues of these substrates, the respective Ki's are as follows: (2RS)-2-benzyl-4-(3-methoxyphenyl)-4-oxobutanoic acid, 180 +/- 40 microM; (2RS)-5-benzamido-2-benzyl-4-oxopentanoic acid (V), 48 +/- 7 microM; (2RS)-2-benzyl-5-(carbobenzoxyglycinamido)-4-oxopentanoic acid (IX), 9 +/- 0.1 microM. For the alcohols derived by reduction of each of these ketones, Ki's are as follows: (2RS,4RS)-2-benzyl-4-(3-methoxyphenyl)-4-hydroxybutanoic acid, 190 +/- 10 microM; (2RS,4RS)-5-benzamido-2-benzyl-4-hydroxybutanoic acid (IV), 160 +/- 62 microM; (2RS,4RS)-2-benzyl-5-(carbobenzoxyglycinamido)-4-hy droxypentanoic acid (XI), 600 +/- 100 microM. Ki values for the competitive peptide ketone inhibitors decrease with increasing peptide chain length. This is consistent with the possibility of increased binding interaction between inhibitor and enzyme by simple occupation of additional binding subsites by adding more amino acid residues to the inhibitor. In contrast, the Ki values of the alcohols (competitive or mixed inhibition) increased or remain essentially unchanged with increasing chain length. Increasing the chain length of ketone inhibitor V to give IX decreases Ki by one-fifth. The Ki of ketone IX is also less than 1/30th the Ki of its isosteric peptide and almost 1/70th that of its isosteric alcohol, XI.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-Phosphonopropionic acids inhibit carboxypeptidase A as multisubstrate analogues or transition-state analogues.

A series of phosphonic acid analogues of 2-benzylsuccinate were tested as inhibitors of carboxypeptidase A. The most potent of these, (2RS)-2-benzyl-3-phosphonopropionic acid, had a Ki of 0.22 +/- 0.05 microM, equipotent to (2RS)-2-benzylsuccinate and thus one of the most potent reversible inhibitors known for this enzyme. Lengthening by one methylene group to (2RS)-2-benzyl-4-phosphonobutyric acid increased the Ki to 370 +/- 60 microM. The monoethyl ester (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid was nearly as potent as (2RS)-2-benzyl-3-phosphonopropionic acid, with a Ki of 0.72 +/- 0.3 microM. The sulphur analogue of the monoethyl ester, 2-ambo-P-ambo-2-benzyl-3-(O-ethylthiophosphono)propionic acid, had a Ki of 2.1 +/- 0.6 microM, nearly as active as (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid. These phosphonic acids and esters could be considered to be multisubstrate inhibitors of carboxypeptidase A by virtue of their structural analogy with 2-benzylsuccinate. Alternatively, the tetrahedral hybridization at the phosphorus atom suggests that they could be mimicking a tetrahedral transition state for the enzyme-catalysed hydrolysis of substrate.     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfamide derivatives as transition state analogue inhibitors for carboxypeptidase A

3-Phenyl-2-sulfamoyloxypropionic acid (2), 2-benzyl-3-sulfamoylpropionic acid (3), and N-(N-hydroxysulfamoyl)phenylalanine (5) have been synthesized and evaluated as inhibitors for carboxypeptidase A (CPA) to find that they inhibit the enzyme competitively with the Ki values in the microM range, suggesting that their binding modes to CPA are analogous to each other, and resemble the binding mode of N-sulfamoylphenylalanine (1) that has been established by the X-ray crystallographic method to form a complex with CPA in a manner reminiscent of the binding of a transition state in the catalytic pathway. It was concluded thus that they are a new type of transition state analogue inhibitors for CPA. (R)-N-Hydroxy-N-sulfamoyl-beta-phenylalanine (8) was shown to be also a potent CPA inhibitor (Ki = 39 microM), the high potency of which may be ascribed to the involvement of the hydroxyl in the binding of CPA, most likely forming bidentate coordinative bonds to the zinc ion in CPA together with the sulfamoyl oxygen atom.     

Mechanism of inactivation of human leukocyte elastase by a chloromethyl ketone: kinetic and solvent isotope effect studies

The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)     

A metabolite of aspartame inhibits angiotensin converting enzyme

Aspartame (L-aspartyl-L-phenylalanine methyl ester, is a widely used artificIal sweetener. In humans and other animals aspartame is initially hydrolyzed to L-aspartyl-L-phenylalanine by intestinal esterases. L-Aspartyl-L-phenylalanine inhibits angiotensin converting enzyme purified from rabbit lungs with a Ki of 11 +/- 2 microM, equipotent to the IC50 of 12 microM for 2-D-methyl-succinyl-L-proline which has been reported to be an orally active antihypertensive agent in rats. Thus the possibility exists that L-aspartyl-L-phenylalanine inhibits angiotensin converting enzyme in humans consuming large quantities of aspartame. Both aspartame itself and the diketopiperazine formed from it, 3-carboxymethyl-6-benzyl-2,5-diketopiperazine, are weak inhibitors with Ki's greater than 1 mM.     

Inhibition of serine proteases by peptidyl fluoromethyl ketones

We have synthesized peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases alpha-chymotrypsin and porcine pancreatic elastase. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the gamma-OH group of the active site serine to form a stable hemiacetal [Lowe, G., & Nurse, D. (1977) J. Chem. Soc., Chem. Commun., 815; Chen, R., Gorenstein, D.G., Kennedy, W.P., Lowe, G., Nurse, D., & Schultz, R.M. (1979) Biochemistry 18, 921; Shah, D.O., Lai, K., & Gorenstein, D.G. (1984) J. Am. Chem. Soc. 106, 4272]. 19F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF3 and Ac-ambo-Phe-CF3 clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrate or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of Ki concomitant with the change from a dipeptide analogue to a tetrapeptide analogue (Ac-Pro-ambo-Ala-CF3, Ki = 3 X 10(-3) M; Ac-Ala-Ala-Pro-ambo-Ala-CF3, Ki = 0.34 X 10(-6) M) correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues [Bartlett, P.A., & Marlowe, C.K. (1983) Biochemistry 22, 4618; Thompson, R.C. (1973) Biochemistry 12, 47].(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions

The first inhibition study of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV and the gamma-class enzyme from another archaeon, Methanosarcina thermophila (Cam) with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, sulfate, etc., are also provided for comparison. The best Cab anion inhibitors were thiocyanate and hydrogen sulfide, with inhibition constants in the range of 0.52-0.70 mM, whereas cyanate, iodide, carbonate, and nitrate were weaker inhibitors (Ki's in the range of 7.8-13.2 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably, whereas the CA substrate bicarbonate, or other anions, such as bromide, nitrite, bisulfite, or sulfamate behave as weak inhibitors (Ki in the range of 40-45 mM). It is interesting to note that the metal poison, coordinating anions cyanide and azide are also rather weak Cab inhibitors (Ki in the range of 27-55 mM), whereas sulfamide is a very weak Cab inhibitor (Ki of 103 mM), although it strongly inhibits Cam (Ki of 70 microM). Surprisingly, phenylboronic and phenylarsonic acids, which have been investigated for the inhibition of all these CAs for the first time, showed very weak activity against the alpha-CA isozymes, but were effective Cab and Cam inhibitors. The best Cab inhibitors were just these two compounds (Ki's of 0.20-0.33 mM), whereas the best Cam inhibitor was sulfamic acid (Ki of 96 nM). These major differences of behavior between the diverse CAs investigated here toward anion inhibitors can be difficultly explained considering the convergent evolution of so diverse enzymes for the binding and turnover of small molecules such as carbon dioxide and anions.     

Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones

The mechanism of inactivation of serine proteinases by peptide halomethyl ketone inhibitors was studied through the inhibition of trypsin with a series of model peptide ketones (Lys-Ala-LysCH2X). In this series, X is a poor leaving group with increasing electron-withdrawing capacity (X = H, CH2CO2CH3, COCH3, OCOCH3, and F), and as expected, the peptide ketones are reversible, competitive inhibitors of trypsin. The strength of binding of these inhibitors to trypsin increases with the electron-withdrawing ability of X, indicating that the inhibition constant Ki obtained is a measure of reversible hemiketal formation between the inhibitor ketone carbonyl group and the hydroxyl group of the active site serine. A Hammett plot of -log Ki vs. sigma I, the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawing power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the reversible binding involves hemiketal formation, which is followed by alkylation of the enzyme.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.     

Nitro as a novel zinc-binding group in the inhibition of carboxypeptidase A

2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (K(i)=0.15 microM). X-ray crystallography discloses that the nitro group well mimics the transition state occurred in the hydrolysis catalyzed by CPA, that is, an O,O'-bidentate coordination to the zinc ion and the two respective hydrogen bonds with Glu-270 and Arg-127. Because the nitro group is a planar species, we proposed (R)-2-benzyl-3-nitropropanoic acid as a pseudo-transition-state analog inhibitor against CPA.     

Selective phosphinate transition-state analogue inhibitors of the protease of human immunodeficiency virus

The phosphinic acid isosteres of di-, tetra- and hexapeptides containing a hydrophobic amino acid side chains at the P1-P'1 positions are powerful inhibitors of Human Immunodeficiency Virus protease. Ki's ranged from 0.4 nM to 26 microM at pH 6.5 and were lower at pH 4.5. The compounds showed no activity against trypsin, weak activity against renin at pH 6.5, moderate activity against pepsin at pH 2.0 (Ki values in the microM range) and substantial activity against cathepsin D at pH 3.5 (Ki values from 9 to 300 nM).     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

Optically active 2-benzyl-3-methanesulfinylpropanoic acid: Synthesis and evaluation as inhibitors for carboxypeptidase A

All four possible stereomers of 2-benzyl-3-methanesulfinylpropanoic acid were synthesized and evaluated as inhibitors for carboxypeptidase A to find that the isomer having the (2S,4S)-configuration is most potent followed by isomers of (2R,4S)- and (2S,4R)-configurations. The stereochemical preferences shown by the isomers of the inhibitor in binding to the enzyme suggest that the sulfoxide oxygen in the inhibitor fails to ligate the active site zinc ion but may form a hydrogen bond with the guanidinium moiety of Arg-127 like the carbonyl oxygen of scissile peptide bond of oligopeptide substrate of the enzyme does. It may thus be inferred that a sulfoxide moiety may serve as an isosterer of a carboxamide moiety.     

Inhibition kinetics of acetylcholinesterase with fluoromethyl ketones

A series of trifluoromethyl ketones that reversibly inhibit acetylcholinesterase and pseudocholinesterase were synthesized. By analogy to chymotrypsin and on the basis of data reported here, we propose that the active-site serine adds to the ketone to form an ionized hemiketal. The compound (5,5,5-trifluoro-4-oxopentyl)trimethylammonium bicarbonate (1) inhibits acetylcholinesterase with Ki = 0.06 X 10(-9)M and pseudocholinesterase with Ki = 70 X 10(-9)M. Replacement of the nitrogen of 1 by carbon (compound 2) increases Ki for 1 200-fold for acetylcholinesterase but does not significantly alter Ki for pseudocholinesterase. The Ki for the methyl ketone corresponding to 2 is 2 X 10(-4)M for both enzymes, as compared with 12 X 10(-9)M for the trifluoromethyl ketone (acetylcholinesterase). For both enzymes, a linear decrease in log Ki with decreasing pK of the inhibitor hydrate was observed with ketones containing from 0 to 3 fluorines. We attribute this effect to the stabilization of the hemiketal oxyanion. The reduction of the pK of the hemiketal by the trifluoromethyl group is an important contributing factor to the low Ki of trifluoromethyl ketones. The inhibition of acetylcholinesterase by tetramethylammonium chloride and trifluoroacetone was compared to the inhibition by 1, which is a composite of the two smaller inhibitors. The entropic advantage of combining the smaller inhibitors into one molecule is 1.1 X 10(3)M. Inhibitors with Ki less than or equal to 70 X 10(-9) M are slow binding (Morrison, 1982; Morrison & Walsh, 1988). The kinetic data do not require formation of a noncovalent complex prior to formation of the ketal, although such a complex(es) cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Choline transport in Fusarium graminearum A 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).     

Inhibition of cathepsin B by peptidyl aldehydes and ketones: slow-binding behavior of a trifluoromethyl ketone

Inhibition of the cysteine proteinase cathepsin B by a series of N-benzyloxycarbonyl-L-phenylalanyl-L-alanine ketones and the analogous aldehyde has been investigated. Surprisingly, whereas the aldehyde was found to be almost as potent a competitive reversible inhibitor as the natural peptidyl aldehyde, leupeptin, the corresponding trifluoromethyl ketone showed comparatively weak (and slow-binding) reversible inhibition. Evaluation of competitive hydration and hemithioketal formation in a model system led to a structure-activity correlation spanning several orders of magnitude in both cathepsin B inhibition constants (Ki) and model system equilibrium data (KRSH,apparent).     

Enzymatic synthesis and inhibitory characteristics of tartronate semialdehyde phosphate

The immediate product of the pyruvate kinase catalyzed phosphorylation of beta-hydroxypyruvate is the enol of tartronate semialdehyde phosphate (TSP). The reaction has the same pH profile as that for the phosphorylation of pyruvate with pK's of 8.2 and 9.7 observed in H2O. This enol tautomerizes in solution to the aldehyde, which in turn becomes hydrated. 31P NMR spectra indicate that the enol resonates approximately 1 ppm upfield from the hydrated aldehyde. By following the tautomerization spectrophotometrically at 240 nm, we have found it to be independent of pH (0.2 min-1 below pH 6 in water), except that it is 2-fold slower above the pK of the phosphate group (6.3 in H2O and 6.7 in D2O). It is 3.6-fold slower in D2O. When this TSP is reduced with NaBH4, approximately 50% of the product is D-2-phosphoglyceric acid (substrate for enolase). Thus, while the immediate product of the phosphorylation rection is the enol of TSP, the eventual product is D,L-TSP. Both the enol and the aldehyde forms of TSP were found to be potent inhibitors of yeast enolase with apparent Ki values of 100 nM and 5 microM, respectively. However, since the aldehyde form is 95-99% hydrated [Stubbe, J., & Abeles, R. (1980) Biochemistry 19, 5505], the true Ki for the aldehyde species is 50-250 nM. The enol of TSP shows slow binding behavior, as expected for an intermediate analogue, with a t1/2 for this process of approximately 15 s (k = 0.046 s-1) and an initial Ki of approximately 200 nM.     

2-Bromo- and 16-bromo-estrogens and related compounds: potential inhibitors for both estradiol 2- and 16 alpha-hydroxylases in rat liver microsomes

Kinetic studies of inhibition of estradiol 2- and 16 alpha-hydroxylase activities in male rat liver microsomes with 2-bromoestrogens, 4-bromo-estrone (4-BrE1), 16 alpha- and 16 beta-bromoestrones and 16 beta-acetylthioestrone (16-AcSE1) were carried out. 2-Bromoestrogens and 4-BrE1 nonspecifically blocked the two enzyme activities in a competitive manner, and 2-bromo-estradiol (2-BrE2) was the most potent inhibitor for the two hydroxylases among the 2- and 4-bromo steroids. Kinetic data, the apparent Ki's for the inhibitors and the apparent Km's for the substrate E2 in the assay, demonstrate that the A-ring bromo steroids are potent inhibitors for the two enzymes (Ki/Km ranging from 0.28 to 0.48 for the 2-hydroxylation and ranging from 0.26 to 0.49 for the 16 alpha-hydroxylation). In contrast, 16-bromoestrones and 16-AcSE1 non-competitively inhibited the 2-hydroxylation (Ki = ca. 70 microM) while the other was competitively blocked by them (Ki/Km ranging from 0.24 to 0.30). These 16-substituted steroids were very potent inhibitors for the 16 alpha-hydroxylase rather than the 2-hydroxylase and preferentially blocked the former.