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.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.     

Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes

The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.     

Differential inhibition of tryptophan synthase and of tryptophanase by the two diastereoisomers of 2,3-dihydro-L-tryptophan. Implications for the stereochemistry of the reaction intermediates

Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.     

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

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

The use of carba(dethia)-coenzyme A (CH2CoA) derivatives for mechanistic investigations

A novel class of acyl-coenzyme A analogues has been synthesized in which the sulphur atom is replaced by methylene. In contrast to their natural thiolester counterparts these acyl-CH2CoA analogues are stable to hydrolysis. They are good substrates for several enzymes that do not attack the thiolester group (carboxylases, mutases, dehydrogenases, epimerases, etc.) and potent inhibitors for most enzymes that do so. Some of the new insights gained by the use of acyl-CH2CoA are discussed in terms of enzymatic mechanisms.     

Quantitative binding models for CYP2C9 based on benzbromarone analogues

The cytochrome P450 (CYP) isoforms involved in xenobiotic metabolism are enzymes whose substrate selectivity remains difficult to predict due to wide specificity and dynamic protein-substrate interactions. To uncover the determinants of specificity for cytochrome CYP2C9, a novel library of benzbromarone (bzbr) inhibitors was used to reevaluate its pharmacophore. CoMSIA was used with the bzbr ligands to generate both quantitative binding models and three-dimensional contour plots that pinpoint predicted interactions that are important for binding to 2C9. Since this class of compounds is more potent than any other toward 2C9, the small molecule properties deemed most ideal by the software were used to address protein-ligand interactions using new mutagenesis and structural data. Nine new bzbr analogues provide evidence that specific electrostatic and hydrophobic interactions contribute the most to 2C9's specificity. Three of the new analogues are better isosteres of bzbr that contain bulky groups adjacent to the phenol and have increased pK(a) values. These ligands test the hypothesis that anionic substrates bind with higher affinity to 2C9. Since they have higher affinity than the previous nonacidic analogues, the importance of bulky groups on the phenol ring appears to have been underestimated. CoMSIA models predict that these bulky groups are favorable for their hydrophobicity, while a negative charge is favored at the ketone oxygen rather than the phenol oxygen. The overlap of this ketone with electronegative groups of other 2C9 substrates suggests they act as key positive charge acceptors.     

Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent 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.     

Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum

The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate. For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3. All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3'. This agrees well with the occupancies of these sites by these residues in type I collagen. However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen. Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen. In general, the class II enzymes have a broader specificity than the class I enzymes. However, they are much less active toward sequences containing Hyp in subsites P1 and P3'. Thus, the two classes of collagenases have similar but complementary sequence specificities. This accounts for the ability of the two classes of enzymes to synergistically digest collagen.     

Bradykinin analogues with beta-amino acid substitutions reveal subtle differences in substrate specificity between the endopeptidases EC 3.4.24.15 and EC 3.4.24.16.

The closely related zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) cleave many common substrates, including bradykinin (BK). As such, there are few substrate-based inhibitors which are sufficiently selective to distinguish their activities. We have used BK analogues with either alanine or beta-amino acid (containing an additional carbon within the peptide backbone) substitutions to elucidate subtle differences in substrate specificity between the enzymes. The cleavage of the analogues by recombinant EP24.15 and EP24.16 was assessed, as well as their ability to inhibit the two enzymes. Alanine-substituted analogues were generally better substrates than BK itself, although differences between the peptidases were observed. Similarly, substitution of the four N-terminal residues with beta-glycine enhanced cleavage in some cases, but not others. beta-Glycine substitution at or near the scissile bond (Phe5-Ser6) completely prevented cleavage by either enzyme: interestingly, these analogues still acted as inhibitors, although with very different affinities for the two enzymes. Also of interest, beta-Gly8-BK was neither a substrate nor an inhibitor of EP24.15, yet could still interact with EP24.16. Finally, while both enzymes could be similarly inhibited by the D-stereoisomer of beta-C3-Phe5-BK (IC50 approximately 20 microM, compared to 8 microM for BK), EP24.16 was relatively insensitive to the L-isomer (IC50 12 approximately microM for EP24.15, >40 microM for EP24.16). These studies indicate subtle differences in substrate specificity between EP24.15 and EP24.16, and suggest that beta-amino acid analogues may be useful as templates for the design of selective inhibitors.     

Peptide inhibitors of Streptomyces dd-carboxypeptidases

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms.     

Recognition and resistance in TEM beta-lactamase

Developing antimicrobials that are less likely to engender resistance has become an important design criterion as more and more drugs fall victim to resistance mutations. One hypothesis is that the more closely an inhibitor resembles a substrate, the more difficult it will be to develop resistant mutations that can at once disfavor the inhibitor and still recognize the substrate. To investigate this hypothesis, 10 transition-state analogues, of greater or lesser similarity to substrates, were tested for inhibition of TEM-1 beta-lactamase, the most widespread resistance enzyme to penicillin antibiotics. The inhibitors were also tested against four characteristic mutant enzymes: TEM-30, TEM-32, TEM-52, and TEM-64. The inhibitor most similar to the substrate, compound 10, was the most potent inhibitor of the WT enzyme, with a K(i) value of 64 nM. Conversely, compound 10 was the most susceptible to the TEM-30 (R244S) mutant, for which inhibition dropped by over 100-fold. The other inhibitors were relatively impervious to the TEM-30 mutant enzyme. To understand recognition and resistance to these transition-state analogues, the structures of four of these inhibitors in complex with TEM-1 were determined by X-ray crystallography. These structures suggest a structural basis for distinguishing inhibitors that mimic the acylation transition state and those that mimic the deacylation transition state; they also suggest how TEM-30 reduces the affinity of compound 10. In cell culture, this inhibitor reversed the resistance of bacteria to ampicillin, reducing minimum inhibitory concentrations of this penicillin by between 4- and 64-fold, depending on the strain of bacteria. Notwithstanding this activity, the resistance of TEM-30, which is already extant in the clinic, suggests that there can be resistance liabilities with substrate-based design.     

Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.     

An episulfide cation (thiiranium ring) trapped in the active site of HAV 3C proteinase inactivated by peptide-based ketone inhibitors

We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.     

Collagenase enzymes from Clostridium: characterization of individual enzymes.

Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized. The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00. Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose. The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity. At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000. The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain. All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive. Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.     

Inhibition studies of soybean trypsin-like enzyme

The results of inhibition studies of soybean trypsin-like enzyme (STLE) by substrate analogues (derivative of arginine) suggested that a net negative charge exists at or near the substrate binding region of the enzyme. On hydrolysis of substrates, this negative charge seems to repel the products from the substrate binding region and facilitate the turn-over of substrates. From the data on inhibition by various amidines, guanidines, and amines, some information about the structure of the hydrophobic binding pocket of STLE was obtained. The inactivation of STLE by irreversible inhibitors, diisopropylfluorophosphate (DFP) and tosyl-lysine chloromethyl ketone (Tos-Lys-CH2Cl), was decreased by competitive inhibitors. This means that these irreversible inhibitors bind with residues at the substrate binding region, probably serine and histidine residues, respectively.