Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors

The peptidyl trifluoromethyl ketones Ac-Phe-CF3 (1) and Ac-Leu-Phe-CF3 (2) are inhibitors of chymotrypsin. They differ in Ki (20 and 2 microM, respectively) as well as in their kinetics of association with chymotrypsin in that 1 is rapidly equilibrating, with an association rate too fast to be observed by steady-state techniques, while 2 is "slow binding", as defined by Morrison and Walsh [Morrison, J. F., & Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 202], with a second-order association rate constant of 750 M-1 s-1 at pH 7.0 [Imperiali, B., & Abeles, R. (1986) Biochemistry 25, 3760]. The crystallographic structures of the complexes of gamma-chymotrypsin with inhibitors 1 and 2 have been determined in order to establish whether structural or conformational differences can be found which account for different kinetic and thermodynamic properties of the two inhibitors. In both complexes, the active-site Ser 195 hydroxyl forms a covalent hemiketal adduct with the trifluoromethyl ketone moiety of the inhibitor. In both complexes, the trifluoromethyl group is partially immobilized, but differences are observed in the degree of interaction of fluorine atoms with the active-site His 57 imidazole ring, with amide nitrogen NH 193, and with other portions of the inhibitor molecule. The enhanced potency of Ac-Leu-Phe-CF3 relative to Ac-Phe-CF3 is accounted for by van der Waals interactions of the leucine side chain of the inhibitor with His 57 and Ile 99 side chains and by a hydrogen bond of the acetyl terminus with amide NH 216 of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Complex of alpha-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy

A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 microM [Imperiali B., & Abeles, R.H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and alpha-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.     

Inhibition of chymotrypsin by peptidyl trifluoromethyl ketones: determinants of slow-binding kinetics

A series of seven peptidyl trifluoromethyl ketone (TFK) inhibitors of chymotrypsin have been prepared which differ at the P1 and P2 subsites. Inhibition equilibria and kinetics of association and dissociation with chymotrypsin have been measured. The association rate of Ac-Phe-CF3 was measured at enzyme concentrations between 8 nM and 117 microM in order to examine the relation between the ketone/hydrate equilibrium of trifluoromethyl ketones and the "slow binding" by these inhibitors. The association rate decreases at high enzyme concentrations, indicating that TFK ketone is the reactive species and that conversion of TFK hydrate to ketone becomes rate limiting under these conditions. Inhibitors with hydrophobic side chains at P2 bind more tightly but more slowly to chymotrypsin, indicating that formation of van der Waals contacts between the P2 side chain and the His 57 and Ile 99 side chains of chymotrypsin is a relatively slow process. Inhibitor properties were compared to the Michaelis-Menten kinetic constants of a homologous series of peptide methyl ester and peptide amide substrates. Plots of log Ki vs log (kcat/Km) are linear with slopes of 0.65 +/- 0.2, indicating that these inhibitors are able to utilize 65% of the total binding energy between chymotrypsin and its hydrolytic transition state.     

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

Antimalarial effects in mice of orally administered peptidyl cysteine protease inhibitors.

The Plasmodium falciparum cysteine protease falcipain is required for the degradation of hemoglobin by erythrocytic malaria parasites. In prior studies, peptidyl inhibitors of falcipain blocked hemoglobin degradation and development by cultured parasites and one of these compounds, when administered parenterally, cured Plasmodium vinckei-infected mice. We now report an evaluation of orally administered peptidyl inhibitors of falcipain in a mouse malaria model. In studies with a fluoromethyl ketone, orally administered morpholine urea-phenylalanine-homophenylalanine-fluoromethyl ketone delayed the progression of murine malaria. In studies of a new series of vinyl sulfones, a set of related compounds demonstrated marked inhibition of falcipain and of parasite biological activities in vitro. One of these compounds, N-methyl piperazine urea-leucine-homophenylalanine-2-naphthalene vinyl sulfone, cured about 40% of mice when administered orally twice-a-day for four days. Our results suggest that peptidyl inhibitors of falcipain have promise as antimalarial chemotherapeutic agents.     

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).     

The inhibition of human neutrophil elastase and cathepsin G by peptidyl 1,2-dicarbonyl derivatives

Neutrophil elastase and cathepsin G are serine proteases that can damage connective tissue and trigger other pathological reactions. Compounds containing a peptide sequence to impart specificity and bearing an alpha-dicarbonyl unit (alpha-diketone or alpha-keto ester) at the carboxy terminus are potent inhibitors of the neutrophil serine proteases (human neutrophil elastase: R-Val-COCH3, Ki = 0.017 microM; R-Val-COOCH3, Ki = 0.002 microM; human neutrophil cathepsin G: R-Phe-COCH3, Ki = 0.8 microM; R-Phe-COOCH3, Ki = 0.44 microM; R = N-(4-[(4-chlorophenyl)sulfonylaminocarbonyl]phenylcarbonyl)+ ++ValylProlyl).     

Crystallographic analysis of transition-state mimics bound to penicillopepsin: phosphorus-containing peptide analogues.

The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray crystallography and refined to crystallographic agreement factors, R ( = sigma parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahederal transition-state intermediate encountered during aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett, P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55, 6268-6274]. All three of these phosphorus-based inhibitors bind virtually identically in the active site of penicillopepsin in a manner that closely approximates that expected for the transition state [James, M. N. G., Sielecki, A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (approximately 2.4 A) to the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have interpreted this distance and the stereochemical environment of the carboxyl and phosphonate groups in terms of a hydrogen bond that most probably has a symmetric single-well potential energy function. The pro-R oxygen atom is the recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are able to assign a neutral status to Asp213 and a partially negatively charged status to Asp33 with reasonable confidence. Similar very short hydrogen bonds involving the active site glutamic acid residues of thermolysin and carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., & Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduction in mitochondrial membrane potential is an early event in Fas-independent CTL-mediated apoptosis.

Cytotoxic T lymphocytes (CTL) can destroy target cells via the Fas-mediated pathway or the granule-mediated pathway. We used Fas-negative target cells to examine for target-cell reduction in mitochondrial membrane potential (DeltaPsi(m)) induced by intact CTL via the granule-mediated pathway. We find that reduction in DeltaPsi(m) is an early step in Fas-independent CTL killing of target cells that precedes phosphatidyl serine translocation, cytosolic protein release, or loss of plasma membrane integrity. Target-cell reduction in DeltaPsi(m) and cytoplasmic protein release in Fas-independent CTL killing were inhibited by N-carbobenzoxy-Ala-Pro-Phe chloromethyl ketone, but not by caspase inhibitors N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone (z-VAD-fmk) or N-carbobenzoxy-Asp-Glu-Val-Asp fluoromethyl ketone (z-DEVD-fmk). This contrasts with Fas-mediated apoptosis, in which the reduction in DeltaPsi(m) can be inhibited by z-VAD-fmk or z-DEVD-fmk. Assessing the changes in target-cell DeltaPsi(m) can provide for a sensitive and rapid means with which to monitor CTL activity.     

Reaction of Lactobacillus histidine decarboxylase with L-histidine methyl ester

L-Histidine methyl ester inactivates histidine decarboxylase in a time-dependent manner. The possibility was considered that an irreversible reaction between enzyme and inhibitor occurs [Recsei, P. A., & Snell, E. E. (1970) Biochemistry 9, 1492-1497]. We have confirmed time-dependent inactivation by histidine methyl ester and have investigated the structure of the enzyme-inhibitor complex. Upon exposure to either 8 M guanidinium chloride or 6% trichloroacetic acid, unchanged histidine methyl ester is recovered. Formation of the complex involves Schiff base formation, most likely with the active site pyruvyl residue [Huynh, Q. K., & Snell, E. E. (1986) J. Biol. Chem. 261, 4389-4394], but does not involve additional irreversible covalent interaction between inhibitor and enzyme. Complex formation is a two-step process involving rapidly reversible formation of a loose complex and essentially irreversible formation of a tight complex. For the formation of the tight complex, Ki = 80 nM and koff = 2.5 X 10(-4) min-1. Time-dependent inhibition was also observed with L-histidine ethyl ester, L-histidinamide, and DL-3-amino-4-(4-imidazolyl)-2-butanone. No inactivation was observed with glycine methyl ester or histamine. We propose that in the catalytic reaction the carboxyl group of the substrate is in a hydrophobic region. The unfavorable interaction between the carboxylate group and the hydrophobic region facilitates decarboxylation [Crosby, J., Stone, R., & Liehard, G. E. (1970) J. Am. Chem. Soc. 92, 2891-2900]. With histidine methyl ester this unfavorable interaction is no longer present; hence, there is tight binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of sphingomyelin vesicles by addition of a bile salt

The interactions of the bile salt sodium taurocholate (TC) in 50 mM Trizma-HCl buffer and 150 mM NaCl (pH 9) at 37 degrees C with membranes composed of sphingomyelin (SM) were studied by dynamic light scattering, cryogenic transmission electron microscopy (cryo-TEM) and turbidity measurements. Small unilamellar SM vesicles were prepared by extrusion. Below the CMC of TC, taurocholate addition leads to vesicle growth due to incorporation of the taurocholate molecules into the vesicle bilayer. At around half the CMC of the bile salt, the SM vesicles are transformed into SM/TC mixed worm-like micelles, which are visualized by cryo-TEM for the first time. Further increase in the taurocholate concentration leads to the rupture of these structures into small spherical micelles. Interestingly, large non-spherical micelles were also identified for pure taurocholate solutions. Similar threadlike structures have been reported earlier for the bile salt sodium taurodeoxycholate [Rich, A., Blow, D., 1958. Nature 182, 1777; Blow, D.M., Rich, A., 1960. J. Am. Chem. Soc. 82, 3566-3571; Galantini, L., Giglio, E., La Mesa, C., Viorel-Pavel, N., Punzo, F., 2002. Langmuir 18, 2812] and for mixtures of taurocholate and phosphatidylcholate [Ulmius, J., Lindblom, G., Wennerstr?m, H., Johansson, L.B.-A., Fontel, K., S?derman, O., Ardvisson, G., 1982. Biochemistry 21, 1553; Hjelm, R.P., Thiyagarajan, P., Alkan-Onyuksel, H., 1992. J. Phys. Chem. 96, 8653] as determined by various scattering methods.     

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.     

Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase

We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.     

A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane

Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637; Kellenberger, C., Hendrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environment have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637]. Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER). The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechnol. 16, 370-375]. Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates. Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane. In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor. The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells.     

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

HRMAS NMR observation of beta-sheet secondary structure on a water swollen solid support.

In this paper HRMAS NMR was used to investigate whether peptides on a peptidyl resin swollen in aqueous solution can adopt an intramolecular beta-sheet structure. A model peptide YQNPDGSQA, that was previously shown to adopt such a secondary structure in solution, (Blanco et al, J. Am. Chem. Soc., 1993) was grafted onto three different solid supports that swell in aqueous solution to examine the influence of the resin on the structure. Both parameters of resin loading and pH inside the swollen peptidyl resin proved to be important for the physicochemical behaviour of the peptide on the support.     

Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD+, Ca2+, citrate, and isocitrate

The activity of NAD-dependent isocitrate dehydrogenase from bovine heart was inhibited by NADH (apparent Ki about 4.3 microM) and NADPH (Ki about 9.8 microM) at subsaturating substrate concentrations at pH 7.4. The inhibition by NADH or NADPH was reversed competitively by magnesium isocitrate in the presence of ADP, but not without ADP. Reversal of inhibition by NADH or NADPH with respect to NAD+ was competitive or of the linear mixed type depending on whether ADP was absent or present. ADP3- (0.2 mM) increased the Ki(app) for NADPH from 9.8 to 27.1 microM; further addition of Ca2+ (0.2 mM) raised the Ki(app) to 127 microM. For the modification of NADPH inhibition by ADP, S0.5 for Ca2+ was approximately 48 microM. This compares to the Km for Ca2+ of 0.3-1 microM for the activation of the enzyme without NADPH [Denton, R. M., Richards, D. A., & Chin, J. G. (1978) Biochem. J. 176, 899-906; Aogaichi, T., Evans, J., Gabriel, J., & Plaut, G. W. E. (1980) Arch. Biochem. Biophys. 204, 350-360]. ADP did not affect the Ki for NADH. Magnesium citrate, which was about 100-fold more effective as a positive modifier of the enzyme with ADP than without ADP [Gabriel, J. L., & Plaut, G. W. E. (1983) Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2082], reversed competitively the inhibition by NADPH in the presence of ADP, but not without ADP. Magnesium citrate did not reverse NADH inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.