Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition

The synthesis of 10 analogues of pepstatin modified so that statine is replaced by 4-amino-3-hydroxy-3,6-dimethylheptanoic acid (Me3Sta) or 4-amino-3-hydroxy-3-methyl-5-phenylpentanoic acid (Me3AHPPA) residues is reported. Both the 3S,4S and 3R,4S diastereomers of each analogue were tested as inhibitors of the aspartic proteases, porcine pepsin, cathepsin D, and penicillopepsin. In all cases the 3R,4S diastereomer (rather than the 3S,4S diastereomer) of the Me3Sta and Me3AHPPA derivatives was found to be the more potent inhibitor of the aspartic protease (Ki = 1.5-10 nM for the best inhibitors), in contrast to the results obtained with statine (Sta) or AHPPA derivatives, where the 3S,4S diastereomer is the more potent inhibitor for each diastereomeric pair of analogues. The Me3Sta- and Me3AHPPA-containing analogues are only about 10-fold less potent than the corresponding statine and AHPPA analogues and 100-1000-fold more potent than the corresponding inhibitors lacking the C-3 hydroxyl group. Difference NMR spectroscopy indicates that the (3R,4S)-Me3Sta derivative induces conformational changes in porcine pepsin comparable to those induced by the binding of pepstatin and that the (3S,4S)-Me3Sta derivatives do not induce the difference NMR spectrum. These results require that the C-3 methylated analogues of statine-containing peptides must inhibit enzymes by a different mechanism than the corresponding statine peptides. It is proposed that pepstatin and (3S)-statine-containing peptides inhibit aspartic proteases by a collected-substrate inhibition mechanism. The enzyme-inhibitor complex is stabilized, relative to pepstatin analogues lacking the C-3 hydroxyl groups, by the favorable entropy derived when enzyme-bound water is returned to bulk solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological activity of new conformationally restricted analogues of pepstatin.

A new statine derivative, 3-hydroxy-4-amino-5-mercaptopentanoic acid; cysteinylstatine (CySta), was synthesized and used to prepare a series of conformationally restricted analogues of pepstatin (Iva-Val-Val-Sta-Ala-Sta) in which the conformational constraint was introduced via a bis-sulfide connecting the appropriately substituted residues in the P1 and the P3 inhibitor side chains. The precursor peptide, Iva-Cys-Val-CySta-Ala-Iaa, was synthesized and alkylated with a series of dibromoalkanes and alkenes to produce the cyclic structures. This strategy permitted the carbon atom spacing between the P1 and the P3 inhibitor side chains to be systematically varied so as to produce inhibitors with 15-, 16-, and 17-membered ring systems. Additional non-cyclic analogues were synthesized as controls by alkylating the bisthiol intermediates with methyl iodide. The inhibitory potency of the analogues were determined against porcine pepsin and penicillopepsin by using standard enzyme kinetic assays. The cyclic inhibitor were found to be potent inhibitors of both aspartic proteases; inhibitor that contained a trans-2-butene link between the two sulfur atoms was found to be the most potent inhibitor with a Ki less than 1 nM against pepsin and 3.94 nM against penicillopepsin. This series of compounds illustrates a new type of conformational restriction that can be used to probe for the bioactive conformation of peptides.     

Biological activity of aspartic proteinase inhibitors related to pepstatin

We have synthesized eight tripeptide analogs of pepstatin in which both the side-chain and stereochemistry of the novel amino acid statine have been altered. They have been compared to pepstatin for inhibition of pepsin and cathepsin D activity, inhibition of autolysis at pH 4, and inhibition of protein degradation in cultured cells. Effective inhibition of aspartic proteinase activity appears to require the novel amino acid to have a bulky hydrophobic side-chain and the S-configuration at both chiral centers. However, the Cbz-Val-Val-(3S4S)-statine peptide was more effective than pepstatin in cultured cells, and inhibition was also achieved, and in some cases enhanced relative to pepstatin, by its stereoisomers and by tripeptides containing valyl and alanyl analogs of statine.     

Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: correlation of the kinetic parameters with the inhibitor induced conformational changes

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.     

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin) and Pig Pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Inhibition of 1,4-beta-D-xylan xylanohydrolase by the specific aspartic protease inhibitor pepstatin: probing the two-step inhibition mechanism

This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC50 value 3.6 +/- 0.5 microm. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 +/- 1 x 10(-5) and 3.0 +/- 1 x 10(-8) s(-1), respectively. The Ki value for the formation of EI complex was 1.5 +/- 0.5 microm, whereas the overall inhibition constant Ki* was 28.0 +/- 1 nm. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.     

The pH-dependence of the binding of competitive inhibitors to pepsin

1. The pH-dependence of the binding to pepsin of four dipeptide competitive inhibitors is reported. Values of K(i) obtained from equilibrium-dialysis experiments agree closely with those from kinetic measurements. 2. The binding of uncharged N-acyl-dipeptide amides to pepsin is essentially independent of pH from 0.2 to 5.8. Values of K(i) for the corresponding N-acyl-dipeptide acids rise rapidly above pH3.5, and depend on the ionization of a group of apparent pK(a) 3.6. 3. The data indicate that pepsin does not undergo any gross conformation change (at least none that affects binding) over the whole pH range of its catalytic activity. The pH-dependence of the dipeptide acid inhibitors indicates that the acid anions do not bind to pepsin, presumably because of electrostatic repulsion between the inhibitor anion and a negative centre at or near the active site of the enzyme. 4. The binding of all four stereoisomers of N-acetylphenylalanylphenylalanine, of the depside analogues of the l-l- and d-l-compounds and of N-acetylglycyl-l-phenylalanine and N-acetyl-l-phenylalanylglycine was studied at pH2.2. 5. These results throw further light on the binding specificity of pepsin and on the charge nature of the active site of this enzyme.     

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.     

NAD + binding by yeast alcohol dehydro-genase in the presence of pyrazole and a new method for the determination of the enzyme active site concentration

The pyrazole method of Theorell and Yonetani ( Biochem. Z., 338: 573-553, 1963) has been adapted for the determination of the enzyme active site concentration in yeast alcohol dehydrogenase (EC 1.1.1.1). This method cannot be applied indiscriminately to other alcohol dehydrogenases without modification.     

Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles

Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindstr?m, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase

1. Pig heart lactate dehydrogenase is inhibited by addition of one equivalent of diethyl pyrocarbonate. The inhibition is due to the acylation of a unique histidine residue which is 10-fold more reactive than free histidine. No other amino acid side chains are modified. 2. The carbethoxyhistidine residue slowly decomposes and the enzyme activity reappears. 3. The essential histidine residue is only slightly protected by the presence of NADH but is completely protected when substrate and substrate analogues bind to the enzyme-NADH complex. The protection is interpreted in terms of a model in which substrates can only bind to the enzyme in which the histidine residue is protonated and is thus not available for reaction with the acylating agent. 4. The apparent pK(a) of the histidine residue in the apoenzyme is 6.8+/-0.2. In the enzyme-NADH complex it is 6.7+/-0.2. 5. Acylated enzyme binds NADH with unchanged affinity. The enzyme is inhibited because substrates and substrate analogues cannot bind at the acylated histidine residue in the enzyme-NADH complex.     

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.     

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 kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate

With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme-ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K(I)) for ATTM as 1.0+/-0.2 microM and 10.65 microM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.     

Inhibition of rat liver steroid 5 alpha-reductase by 3-androstene-3-carboxylic acids: mechanism of enzyme-inhibitor interaction

The interactions of a series of newly discovered inhibitors of delta 4-3-oxo-steroid 5 alpha-reductase (SR; EC 1.3.1.30), the 3-androstene-3-carboxylic acids (steroidal acrylates), have been studied by using a solubilized rat liver enzyme preparation. As exemplified by one member of this series, 17 beta-[N,N-diisopropyl-carbamoyl)androst-3,5-diene-3-carboxylic acid (1a), the dead-end inhibition patterns of selected compounds in this class are best evaluated by a linear uncompetitive kinetic model versus either substrate, testosterone (T) or NADPH. These results were interpreted within the context of the preferentially ordered kinetic mechanism for rat liver SR to arise from the association of inhibitor to the binary complex of enzyme and NADP+. This proposed inhibition mechanism was supported by data from double-inhibition experiments implicating the synergistic binding of steroidal acrylate and NADP+ to SR. Further evidence for the preferential formation of this ternary complex was obtained from filtration binding assays with [3H]-1a, where radioligand association to protein was greatly enhanced in the presence of NADP+. The amount of [3H]-1a binding to protein was proportional to the specific activity of SR in the enzyme preparations, and the estimated dissociation constant from binding data by Scatchard analysis (Kd = 25 nM) was comparable to the inhibition constants estimated for SR activity (Ki = 12-26 nM). From the pH profile for inhibition of the solubilized liver SR with 1a, it is proposed that the anion of the steroidal acrylate (pK1 = 4.7 +/- 0.2) is the active inhibitory species, coordinating to a protonated active site functionality (pK2 = 7.5 +/- 0.1). On the basis of data from similar experiments with structural analogues of 1a, the determinants for binding recognition and inhibitory potency are compared to structural features of the putative enzyme-bound intermediate states. These compounds represent a potential therapeutic alternative in the treatment of 5 alpha-dihydrotestosterone specific androgen dependent disease states.     

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.     

Synthesis of A 3-oxo-4(S)-amino acid analog of pepstatin. A new inhibitor of carboxyl (acid) proteases

A ketone analog of pepstatin, in which the 3S hydroxyl group is oxidized to a ketone group, has been synthesized and shown to be a potent inhibitor of pepsin. Kinetics of inhibition of pepsin provide evidence that the ketone pepstatin analog binds to pepsin differently than pepstatin. The relationship of these complexes to crystal complexes of pepstatin-carboxyl proteases is discussed.