Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.     

Interrelationship of active and latent secreted human cathepsin B precursors.

Two high-Mr forms of cathepsin B have been described previously, both of which are stable at alkaline pH, in contrast with the lysosomal proteinase. One form is latent and activated by pepsin treatment; the other form is active as measured with synthetic substrates. In the present study it was shown that the two forms are indistinguishable on the basis of molecular size as determined by gel-filtration chromatography or sodium dodecyl sulphate/polyacrylamide-gel electrophoresis followed by immunoblotting. Both forms lose their alkali-stability upon exposure to Hg2+, and after Hg2+ treatment the latent form becomes immuneprecipitable by an antiserum that reacts only with denatured cathepsin B. Lysosomal cathepsin B is bound by the plasma proteinase inhibitor alpha 2-macroglobulin, a process that requires proteolytic cleavage of the inhibitor. In contrast, the stable active form of cathepsin B is not bound by this inhibitor unless this enzyme is first destabilized by Hg2+ treatment. These results indicate that cathepsin B exists in three different states of activity, completely latent, partially active and fully proteolytically active. To exhibit true endopeptidase activity it seems that the enzyme must be in an alkali-unstable form.     

Antioxidative function and substrate specificity of NAD(P)H-dependent alkenal/one oxidoreductase. A new role for leukotriene B4 12-hydroxydehydrogenase/15-oxoprostaglandin 13-reductase

There are several known routes for the metabolic detoxication of alpha,beta-unsaturated aldehydes and ketones, including conjugation to glutathione and reduction and oxidation of the aldehyde to an alcohol and a carboxylic acid, respectively. In this study, we describe a fourth class of detoxication that involves the reduction of the alpha,beta-carbon=carbon double bond to a single bond. This reaction is catalyzed by NAD(P)H-dependent alkenal/one oxidoreductase (AO), an enzyme heretofore known as leukotriene B4 12-hydroxydehydrogenase, 15-oxoprostaglandin 13-reductase, and dithiolethione-inducible gene-1. AO is shown to effectively reduce cytotoxic lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) (k(cat) = 4.0 x 10(3) min(-1); k(cat)/K(m) = 3.3 x 10(7) min(-1) M(-1)) and acrolein (k(cat) = 2.2 x 10(2) min(-1); k(cat)/K(m) = 1.5 x 10(6) min(-1) M(-1)) and common industrial compounds such as ethyl vinyl ketone (k(cat) = 9.6 x 10(3) min(-1); k(cat)/K(m) = 8.8 x 10(7) min(-1) M(-1)) and 15-oxoprostaglandin E1 (k(cat) = 2.4 x 10(3) min(-1); k(cat)/K(m) = 2.4 x 10(9) min(-1) M(-1)). Furthermore, transfection of human embryonic kidney cells with a rat liver AO expression vector protected these cells from challenge with HNE. The concentration of HNE at which 50% of the cells were killed after 24 h increased from approximately 15 microM in control cells to approximately 70 microM in AO-transfected cells. Overexpression of AO also completely abolished protein alkylation by HNE at all concentrations tested (up to 30 microM). Thus, we describe a novel antioxidative activity of a previously characterized bioactive lipid-metabolizing enzyme that could prove to be therapeutically or prophylactically useful due to its high catalytic rate and inducibility.     

Inhibition of cathepsin K with lysosomotropic macromolecular inhibitors

Cathepsin K is the major enzyme responsible for the degradation of the protein matrix of bone and probably for the destruction of articular cartilage in rheumatoid arthritis joints. These processes occur mainly in the resorption lacuna and within the lysosomal compartment. Here, we have designed, synthesized, and evaluated new lysosomotropic (water-soluble) polymer-cathepsin K inhibitor conjugates. In particular, we characterized the relationship between conjugate structures and their activity to inhibit cathepsins K, B, L, and papain. A potent selective cathepsin K inhibitor, 1,5-bis(N-benzyloxycarbonylleucyl)carbohydrazide, was modified to 1-(N-benzyloxycarbonylleucyl)-5-(phenylalanylleucyl)carbohydrazide (I) to facilitate polymer conjugation. It was conjugated to the polymer chain termini of two water-soluble polymers [alpha-methoxy poly(ethylene glycol), abbreviated as mPEG-I; semitelechelic poly[N-(2-hydroxypropyl)methacrylamide], abbreviated as ST-PHPMA-I]. The conjugation of inhibitor I to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer side chains was accomplished via either a Gly-Gly spacer (PHPMA-GG-I) or with no spacer between I and the copolymer backbone (PHPMA-I). Kinetic analysis revealed that free inhibitor I possessed an apparent second-order rate constant against cathepsin K (k(obs)/[I] = 1.3 x 10(6) M(-1) s(-1)) similar to that of unmodified 1,5-bis(Cbz-Leu) carbohydrazide, while I conjugated to the chain termini of mPEG and ST-PHPMA-COOH had slightly lower values (about 5 x 10(5) M(-1) s(-1)). The k(obs)/[I] values for I attached to the side chains of HPMA copolymers (PHPMA-GG-I and PHPMA-I) were about 3 x 10(4) M(-1) s(-1). When tested against cathepsin L, inhibitor I and all its polymer conjugates produced k(obs)/[I] values 1-2 orders of magnitude less than those determined for cathepsin K, while for cathepsin B and papain, the values were 2-4 orders of magnitude lower. The ability of mPEG-I and ST-PHPMA-I to inhibit cathepsin K activity in synovial fibroblasts was also evaluated. Both polymer-bound inhibitors were internalized by endocytosis and were ultimately trafficked to the lysosomal compartment. ST-PHPMA-I was internalized faster than mPEG-I. The inhibitory activity in the synovial fibroblast assay correlated with the rate of internalization.     

Cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from mouse and guinea pig liver Golgi membranes during the acute phase response

1. Rat Gal beta 1-4GlcNAc alpha 2-6sialyltransferase (E.C. 2.4.99.1) is released from Golgi membranes by cleavage of a portion of the enzyme containing the active site from a membrane anchor; this effect was most dramatic during the acute phase response. The enzyme that cleaved sialyltransferase had the properties of cathepsin D was most active at pH 5.6 and was likely of lysosomal origin (Lammers and Jamieson, 1988). 2. The acute phase response of sialyltransferase in mouse and guinea pig was previously found to differ from that in the rat. Release of sialyltransferase from mouse and guinea pig Golgi membranes has now been studied in order to make a comparison with the rat system. 3. Maximum release of sialyltransferase from mouse and guinea pig Golgi occurred at pH 4.6 and 5.2, respectively; like the rat a cathepsin D-like proteinase was responsible for release of both enzymes. 4. Immunoblot analysis showed that membrane-bound rat and mouse sialyltransferase had Mr 49,000, whereas the guinea pig enzyme had Mr 42,000. The released form of the rat enzyme had Mr 42,000, but released forms of mouse and guinea pig enzymes had Mr 38,000 suggesting a different cleavage site for these two enzymes compared to the rat enzyme.     

Enzyme inhibition assays using fluorescence correlation spectroscopy: a new algorithm for the derivation of kcat/KM and Ki values at substrate concentrations much lower than the Michaelis constant

A new mathematical formalism is deduced which allows for the calculation of the k(cat) over K(M) ratio based on measurements of the enzyme kinetics with substrate concentrations much lower than K(M). The equations are also applied on the action of an inhibitor on enzyme activity yielding the binding constant, K(i), of an inhibitor molecule. For practical evaluation of the new theoretical approach, the enzymatic reaction of CD45 phosphatase was used as a well-characterized model system with known inhibitors for testing the K(i) value determination scheme. The k(cat)/K(M) ratio was calulated to be 4.7 x 10(5) M(-)(1) s(-)(1), the K(i) of the inhibitor molecule PKF52-524 was estimated to be (1-2) x 10(-)(7) M and the association rate of the inhibitor PKF52-524 to CD45 phosphatase was estimated to be 59 M(-)(1) s(-)(1).     

Anomalous pH dependence of the reactions of carbenicillin and sulbenicillin with Bacillus cereus beta-lactamase I. Influence of the alpha-substituent charge on the kinetic parameters

The pH dependence of k(cat) for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of carbenicillin(VI), which differs from benzylpenicillin (I) in having a carboxylic moiety alpha to the phenyl ring, exhibits a profile consistent with a model in which the alpha-COOH and alpha-COO forms of the ES complex turn over with respective rate constants of 2152 s(-1) and 384 s(-1). The pK(a)(app) for the alpha-COOH is shifted from 3.2 in solution to 6.1 in the ES complex. The normalized k(cat)/K(m) vs. pH profile for VI is not superimposable on that of I, indicating that both the neutral and anionic forms of the carboxyl moiety of VI combine with the enzyme to give the first irreversibly formed complex, presumably the acyl-enzyme. Quantitative accord with the kinetic data is achieved only through fitting to a model where kinetically significant proton transfer in the ES complex is permitted. The second-order rate constants for the reaction of the enzyme with the alpha-COOH and alpha-COO forms of VI are 2.2 x 10(8) M(-1) s(-1) and 3.8 x 10(6) M(-1) s(-1), respectively. The high value for the alpha-COOH form suggests that this reaction may be in part diffusion controlled. This conjecture is borne out by the observation that the sensitivity of k(cat)/K(m) to eta(rel) decreases with increasing pH for VI, whereas this sensitivity is pH independent for I. These conclusions are further supported by the results of a kinetic investigation of the pH dependence of sulbenicillin (VII) where an alpha-SO3H replaces the alpha-COOH of VI. The strongly acidic sulfonic acid moiety of VII is fully ionized throughout nearly the entire pH range of interest, and its kinetics, as a function of pH, are very similar to those observed and calculated for the alpha-COO form of VI. Solvent deuterium kinetic isotope effects are reported for k(cat) and k(cat)/K(m) for both VI and VII.     

Human cathepsin X: A cysteine protease with unique carboxypeptidase activity.

Cathepsin X is a novel cysteine protease which was identified recently from the EST (expressed sequence tags) database. In a homology model of the mature cathepsin X, a unique three residue insertion between the Gln22 of the oxyanion hole and the active site Cys31 was found to be located in the primed region of the binding cleft as part of a surface loop corresponding to residues His23 to Tyr27, which we have termed the "mini-loop". From the model, it became apparent that this distinctive structural feature might confer exopeptidase activity to the enzyme. To verify this hypothesis, human procathepsin X was expressed in Pichia pastoris and converted to mature cathepsin X using small amounts of human cathepsin L. Cathepsin X was found to display excellent carboxypeptidase activity against the substrate Abz-FRF(4NO(2)), with a k(cat)/K(M) value of 1.23 x 10(5) M(-)(1) s(-)(1) at the optimal pH of 5.0. However, the activity of cathepsin X against the substrates Cbz-FR-MCA and Abz-AFRSAAQ-EDDnp was found to be extremely low, with k(cat)/K(M) values lower than 70 M(-)(1) s(-)(1). Therefore, cathepsin X displays a stricter exopeptidase activity than cathepsin B. No inhibition of cathepsin X by cystatin C could be detected up to a concentration of 4 microM of inhibitor. From a model of the protease complexed with Cbz-FRF, the bound carboxypeptidase substrate is predicted to establish a number of favorable contacts within the cathepsin X binding site, in particular with residues His23 and Tyr27 from the mini-loop. The presence of the mini-loop restricts the accessibility of cystatin C as well as of the endopeptidase and MCA substrates in the primed subsites of the protease. The marked structural and functional differences of cathepsin X relative to other members of the papain family of cysteine proteases will be of great value in designing specific inhibitors useful as research tools to investigate the physiological and potential pathological roles of this novel enzyme.     

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.     

Differential inhibition of rat mast cell proteinase I and II by members of the alpha-1-proteinase inhibitor family of serine proteinase inhibitors

Rat mast cell proteinase II (RMCP II) from mucosal mast cells was titrated into rat serum, and the resulting serine proteinase inhibitor (serpin)-enzyme complex was purified by affinity chromatography on anti-RMCP II-Sepharose 4B and by Mono-Q anion-exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCP II-Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) that inhibit RMCP II in rat serum. The kinetic constants characterizing the interaction between RMCP II and RSI and RSII are ka, 2.2 x 10(5) and 1.65 x 10(5) M-1 s-1, respectively; Ki, 3.6 x 10(-10) and 1.0 x 10(-9) M; and kd, 7.9 x 10(-5) and 1.65 x 10(-4) s-1. Amino-terminal sequence analysis indicated that RSI and RSII are distinct, differing at the amino-terminal residues, and are products of the rat Spi-1 locus. Rat mast cell proteinase I (RMCP I) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited.     

Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Designing of substrates and inhibitors of bovine alpha-chymotrypsin with synthetic phenylalanine analogues in position P(1)

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.     

Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.     

Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.     

Protease-specificity of Kunitz inhibitor domain of Alzheimer's disease amyloid protein precursor

The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E. coli containing a synthetic gene encoding the Kunitz domain. The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme. It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M). In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.     

Identification of the primary antimicrobial domains in human neutrophil cathepsin G

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.     

Partial purification and properties of cathepsin G-like proteinase of mouse myeloid leukemia M1 cells

A proteinase extracted with 1M NaCl from particulate fraction of the postnuclear fraction of mouse myeloid leukemia M1 cells was partially purified by Bio-Gel HTP treatment and Sephadex G-75 gel filtration. The apparent molecular mass of the proteinase was 26,000 Da and the isoelectric point was about pH 10. The enzyme activity was inhibited by phenylmethanesulfonylfluoride, chymostatin, and soy-bean trypsin inhibitor. It hydrolysed specifically Suc-Ala2-Pro-Phe-4-methylcoumaryl-4-amide (MCA). NaCl and KCl enhanced several times the activity for Suc-Ala2-Pro-Phe-MCA, but not that for fluorescein-labeled albumin and fibrinogen. These enzymic properties of the major proteinase are similar to those of chymotrypsin and cathepsin G. The role of a cathepsin G-like proteinase in relation to M1 cell differentiation is discussed.