Inhibitor Of Alpha-chymotrypsin Research Articles

Structural Optimization of Photoswitch Ligands for Surface Attachment of α-Chymotrypsin and Regulation of Its Surface Binding

A modified gold surface that allows photoregulated binding of alpha-chymotrypsin has previously been reported. Here the development of this surface is reported, through the synthesis of a series of trifluoromethyl ketones and alpha-keto esters containing the azobenzene group and a surface attachment group as photoswitch inhibitors of alpha-chymotrypsin. All of the compounds are inhibitors of the enzyme, with activity that can be modulated by photoisomerization. The best photoswitch shows a reversible change in IC(50) inhibition constant of >5.3 times on photoisomerization. The trifluoromethyl ketone 1 exhibited excellent photoswitching and was attached to a gold surface in a two-step procedure involving an azide-alkyne cycloaddition. The resulting modified surface bound alpha-chymotrypsin to a degree that could be modulated by UV/Vis irradiation, showing "slow-tight" enzyme binding as observed for inhibitors in solution.

Inhibition of Chymotrypsin by a Complex of Ortho-Vanadate and Benzohydroxamic Acid: Structure of the Inert Complex and Its Mechanistic Interpretation

Serine proteases, like serine beta-lactamases, are rapidly and covalently inhibited by suitably designed phosph(on)ates. The active sites of these enzymes must, therefore, be able to stabilize the pentacoordinated transition states of phosphyl transfer reactions as well as the tetrahedral transition states of acyl transfers. It follows that these enzymes should also be inhibited by molecules capable of generating inert pentacoordinated species. We (J.H.B. and R.F.P.) have previously shown that these enzymes are, in fact, rapidly and reversibly inhibited by 1:1 complexes of vanadate and hydroxamic acids. In this paper, we present the first crystal structure of an acyl transferase inhibited by vanadate. The complex of vanadate and benzohydroxamic acid is a competitive inhibitor of alpha-chymotrypsin with a KI value of 16 muM. In the structure, obtained at a resolution of 1.5 A, the protein is conformationally little different from the apoenzyme. The vanadium, in a distorted octahedral ligand field, is covalently bound to the active site serine oxygen group. One oxgen ligand, presumably anionic, is located in the oxyanion hole. Another is directed roughly in the direction of the acyl transfer leaving group, and a third in the direction of the S2 site. The hydroxamate is bound to vanadium through the hydroxyl oxygen and also, more weakly, through the carbonyl group, to form a five-membered chelate ring. The effect of this chelation is to place the phenyl group of the inhibitor into the important S1 specificity site. The hydroxamate oxygen is directed in line away from the Ser195 Ogamma, approximating the direction of departure of a leaving group in phosphyl transfer. The entire complex can be seen as a reasonable mimic of a phosphyl transfer transition state where the leaving group is extended into the S1 site.

Noncovalent inhibition of the serine proteases, α-chymotrypsin and trypsin by trifluoro(organo)borates

A series of potassium organotrifluoroborates were synthesized. Their stability to hydrolysis was determined in D2O, TRIS and phosphate buffer. It was found that in both D2O and TRIS buffers, these compounds are quite stable, whereas in phosphate buffer rapid hydrolysis occurs. Based on these results, a study was undertaken to determine whether potassium organotrifluoroborates can serve as protease inhibitors. It was found that potassium organotrifluoroborates increased inhibition by at least an order of magnitude over the corresponding boronates. Dixon plots showed that these compounds are reversible competitive inhibitors of alpha-chymotrypsin and trypsin. Based on 19F NMR, we speculate that they inactivate the enzymes as a result of the formation of hydrogen-bonds between fluorine atoms of the inhibitors and the serine protease.

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine alpha-chymotrypsin at 2.1- and 3.0-A resolution, respectively. PMP-C is a potent bovine alpha-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine alpha-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel beta-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P'4, corresponding to residues 26-34) and of an internal segment (residues 15-18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21-25) and the residues 172-175 of alpha-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P'4).

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

The molecular evolutionary history of a winged bean alpha-chymotrypsin inhibitor and modeling of its mutations through structural analyses.

A serine protease inhibitor of the Kunitz-STI (soybean trypsin inhibitor) family, isolated from the legume seeds of winged bean, was found to inhibit chymotrypsin at a 1:2 stoichiometric ratio. When the structure was determined in our laboratory, it was found to form a characteristic beta-trefoil fold, which is also seen in other proteins from distant families and sources. The folding organization divides the protein into three approximately equal subdomains related by a pseudo-threefold axis of symmetry passing parallel to the barrel axis of the trefoil. Following the now established idea that the present-day genes originated from ancestral minigenes through evolution, the origin of the proteins having this beta-trefoil organization is scrutinized using its subdomain motif as the search probe. The results, based mainly on structural analyses, indicate the independent existence of such a motif, mimicking the unknown ancestral protein(s) that might have been distributed in nature, not only by gene duplication, but also by insertion and permutation in other folds. The understanding led to a hypothesis for the possible origin of the Kunitz-STI family. On the basis of this model of evolution, structurally hypervariable regions were located on the protein where mutations could be designed and a broad range of engineering of the protein's activity could be conceived.

A biochemical logic gate using an enzyme and its inhibitor. 1. The inhibitor as switching element.

Molecular-scale logic systems will allow for further miniaturization of information processing assemblies and contribute to a better understanding of brain function. Of much interest are the pertinent biological systems, some of the basic components of which are biomolecular switching elements and enzyme-based logic gates. In this series of accounts, results of investigations are presented on the implementation of an enzyme/inhibitor logic gate operating under the rules of Boolean algebra. In this report (part 1 of the series), consideration is given to the experimental conditions-particularly the irradiation mode-that affect the performance of proflavine as inhibitor of alpha-chymotrypsin. Also, assessments are made on the reversibility of the process involved and the long-term stability of the system. Moreover, using a theoretical conformational analysis of proflavine and its reduction products, detailed features were established regarding their three-dimensional structure, partial charge distribution, and hydrophobicity. Accordingly, an understanding was reached as to the factors affecting the interaction between these compounds and the enzyme. In part 2 of this series, the actual implementation of an AND logic gate will be presented. This gate involves proflavine and a chemically derivatized alpha-chymotrypsin, and its operation relies on the conclusions reached in this report regarding the optimal mode for controlling the inhibitory activity of proflavine.

Refined crystal structure (2.3 A) of a double-headed winged bean alpha-chymotrypsin inhibitor and location of its second reactive site.

The crystal structure of a double-headed alpha-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 A resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6(1)22 with cell parameters a = b = 61.8 A and c = 212.8 A. The final model has a good stereochemistry and a root mean square deviation of 0.011 A and 1.14 degrees from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 A led to an understanding of the mechanism of inhibition of the protein against alpha-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve anti-parallel beta-strands with connecting loops arranged in a characteristic beta-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones.

A novel method for selection of chymotrypsin inhibitors from a phage peptide library.

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Potentially Macrocyclic Peptidyl Boronic Acids as Chymotrypsin Inhibitors.

The possibility of forming a peptide boronate adduct in a serine protease active site that mimics the first tetrahedral intermediate in the peptide hydrolysis mechanism was explored with the complex boronic acid analogs 7, 8-OH, and 8-NH(2)(). In these structures, the P(1) and P(2) residues and the P(1)'-P(3)' residues are connected through the P(2) and P(1)' side chains, to encourage formation of the diester or amide-ester adducts via macrocyclization. These inhibitors were assembled from suitably protected derivatives of 2,4-diaminobutanoic acid or 2,4-diaminopentanoic acid (11), borophenylalanine (12), aspartic acid, malic acid or the substituted malic acid analog 13, and Leu-Arg dipeptide. Stereoselective syntheses were developed for the (S,S)-2,4-diaminopentanoate 11 and for the (S,S)-beta-isobutylmalate 13 derivatives. The complex peptidyl boronates 7 (K(i) = 26 nM) and 8-OH (68 nM) are potent inhibitors of alpha-chymotrypsin; however, the affinity of 7 is neither time- nor pH-dependent, and it is only moderately greater than that found for comparison compounds like 8-H (114 nM), 9 (356 nM), and 10 (219 nM) that cannot cyclize or form a diester adduct.

Esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid as inhibitors of alpha-chymotrypsin: significance of the "aromatic" nature of the novel ester-type coumarin for strong inhibitory activity.

A series of esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid were synthesized and evaluated in vitro for their inhibitory activity toward bovine alpha-chymotrypsin and human leukocyte elastase. Both series behaved as time-dependent inhibitors of alpha-chymotrypsin, but ester-type coumarins were clearly more efficient than the corresponding amides in inactivating the serine proteinase. The best inactivations were observed with "aromatic" esters, in particular with meta-substituted phenyl esters such as m-chlorophenyl 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylate, which appears to be one of the most powerful inactivators of alpha-chymotrypsin yet reported (kinact/KI = 760,000 M-1 S-1 at pH 7.5 and 25 degrees C). Usually, the coumarin derivatives failed to inhibit significantly human leukocyte elastase. As a result, the reported series of aromatic coumarinic esters behaves as a new chemical family of selective alpha-chymotrypsin inhibitors.

Serine Protease Inhibition by Insect Peptides Containing a Cysteine Knot and a Triple-stranded β-Sheet

Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparalled beta-sheet were studied. Their inhibitory effect on two serine proteases (bovine alpha-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong alpha-chymotrypsin inhibitor (Ki = 0.2 nM) and interacts with leukocyte elastase with a Ki of 0.12 microM. The other two peptides, PMP-D2 and HI, interact only weakly with alpha-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both alpha-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a Ki of 3 nM.

Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitors.

A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural L-amino acids (except for cysteine), plus the non-natural L-amino acids ornithine and norleucine, The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN', alpha-chymotrypsin, trypsin, anhydro-alpha-chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to alpha-chymotrypsin, two of which also bind to anhydro-alpha-chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K(i)) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K(i) values. In the case of the alpha-chymotrypsin inhibitors, the peptides with the lowest K(i) (0.1-0.05 mM) were the only peptides that bound to anhydro-alpha-chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that alpha-chymotrypsin and trypsin are inhibited by these cyclic peptides.

5-(Halomethyl)-2-pyranones as irreversible inhibitors of alpha-chymotrypsin.

A series of 5-(halomethyl)-2-pyranones were prepared and assayed as potential mechanism-based inhibitors of chymotrypsin, in order to test whether substitution of a suitable electron-withdrawing group at position 5 in a 2-pyranone can activate the ring for catalytic hydrolysis, as well as form a mechanism-based inhibitor. 3-Benzyl-5-(chloromethyl)-2-pyranone (3) and 3-(1-naphthylmethyl)-5-(chloromethyl)-2-pyranone (4) rapidly and irreversibly inactivate chymotrypsin with high efficiency (I/E = 2.0 for 3 and 1.38 for 4). However, evidence for their formation of an acyl enzyme is lacking, and thus they may simply be active-site alkylating agents. 6-Methyl substitution on 4, or replacement of the chloromethyl of 3 with CF3, prevents inactivation. Since 6-bromo substitution is capable of ring activation, but 5-bromo substitution (3-benzyl-5-bromo-2-pyranone (11)) is not, the 5-position of the pyrone does not appear to affect catalytic hydrolysis.

ChemInform Abstract: Acylamino Boronic Acids and Difluoroborane Analogues of Amino Acids: Potent Inhibitors of Chymotrypsin and Elastase.

AbstractDie Boronate (IV) werden in die Boronsäuren (VII) und die Difluoride (VIII) übergeführt, die als Inhibitoren von Elastase und α‐Chymotrypsin untersucht werden.

ChemInform Abstract: EFFECTS OF PLATINUM COMPLEXES ON CHYMOTRYPSIN

A variety of platinum complexes were evaluated as inhibitors of alpha-chymotrypsin. A standard enzyme assay was used with benzoyl-L-tyrosine ethyl ester as the substrate. Rb2PtBr4, trans-Pt(NH3)2Cl2, and K2PtCl4 were all effective enzyme inhibitors. Pt(Gly-L-Met(Cl2, Pt(Met)(NH3)2Cl, cis-Pt(NH3)2Cl2, and two ethylenediamminedichloroplatinum complexes were weak inhibitors of alpha-chymotrypsin. A surprising finding was the noninhibition of an immobilized preparation of alpha-chymotrypsin by the above inhibitors and by active-site-directed modifying reagents.

Effects of platinum complexes on chymotrypsin

A variety of platinum complexes were evaluated as inhibitors of alpha-chymotrypsin. A standard enzyme assay was used with benzoyl-L-tyrosine ethyl ester as the substrate. Rb2PtBr4, trans-Pt(NH3)2Cl2, and K2PtCl4 were all effective enzyme inhibitors. Pt(Gly-L-Met(Cl2, Pt(Met)(NH3)2Cl, cis-Pt(NH3)2Cl2, and two ethylenediamminedichloroplatinum complexes were weak inhibitors of alpha-chymotrypsin. A surprising finding was the noninhibition of an immobilized preparation of alpha-chymotrypsin by the above inhibitors and by active-site-directed modifying reagents.

Evidence for hemiacetal formation between N-acyl-L-phenylalaninals and .alpha.-chymotrypsin by cross-saturation nuclear magnetic resonance spectroscopy

N-Acetyl-L-phenylalaninal exists predominantly in its hydrated form in aqueous solution, but the aldehyde and not the hydrate is shown by nuclear magnetic resonance (NMR) spectroscopy to be the effective inhibitor of alpha-chymotrypsin. NMR spectroscopy also indicates that the initial alpha-chymotrypsin-N-acetyl-L-phenylalaninal complex is in equilibrium with a hemiacetal formed between the aldehyde and the active site serine residue. The rate of the latter equilibration is slow on the NMR time scale but the hemiacetal can be detected by cross-saturation NMR spectroscopy. N-Benzoyl-L-phenylalaninal is a more potent inhibitor of alpha-chymotrypsin than the N-acetyl derivative and both the formation of the enzyme-inhibitor complex and the hemiacetal are slow on the NMR time scale, but the hemiacetal in the enzyme can be detected by cross-saturation NMR spectroscopy. The N-acyl-L-phenylalaninals also bind to N-methylhistidinyl-57-alpha-chymotrypsin, but clear evidence for hemiacetal formation was not obtained by cross-saturation NMR spectroscopy either because the hemiacetal was not formed or more probably because the rate of dissociation was slow compared with the rate of relaxation of the hemiacetal proton. The dissociation constant of N-benzoyl-L-phenylalaninal to dehydroalaninyl-195-alpha-chymotrypsin was found to be high relative to the dissociation constant to native alpha-chymotrypsin, supporting the NMR evidence that a hemiacetal with the Ser-195 is formed on association of N-benzoyl-L-phenylalaninal with alpha-chymotrypsin.

A soybean trypsin inhibitor. Crystallization and x-ray crystallographic study.

Five trypsin and alpha-chymotrypsin inhibitors which have low molecular weights (ranging from 6800 to 8600) and are present in soybean seeds of the Tracy variety have been isolated and purified, and single crystals which give x-ray diffraction data beyond 3-A spacings have been obtained from one of them. The trypsin inhibitor crystallizes in a monoclinic unit cell of symmetry P2(1) and dimensions a = 25.919(7) A, b = 43.23(1) A, c = 19.905(5) A, and beta = 103.63(2) degrees. The assymmetric unit contains 1 molecule of molecular weight 6800. The crystal, which has been found to be unusually stable to x-radiation, has solvent content of approximately 26% by volume.

A chymotrypsin inhibitor from the parastitic nematode, Oesophagostomum radiatum.

An inhibitor of alpha-chymotrypsin was isolated from O. radiatum by affinity chromatography, as well as by ion-exchange and molecular sieve chromatography. The inhibitor was associated with the cuticle or musculature of the parasite. It completely inhibited the esterolytic activity of chymotrypsin. Molecular sieve chromatography gave an apparent molecular weight of 9700 for the inhibitor alone and 32000 for the complex with chymotrypsin. This suggests reaction in a 1:1 molar ratio. The inhibitor reacted with diisopropyl phosphoryl-chymotrypsin weakly, if at all. Measurement of the dissociation constants for the enzyme-inhibitor complexes gave values of 2.2x10(-9) M for the product of affinity chromatography but less than 2x10(-10) M for all other preparations. Possible explanations of this are discussed.