Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structure of human rhinovirus-14 3C protease (3Cpro) has been determined at 2.3 A resolution and refined to an R factor of 0.22. This cysteine protease folds into two topologically equivalent six-stranded beta barrels and in this sense is similar to trypsin-like serine proteases. However, there are differences in the lengths and positioning of individual beta strands as well as in loops connecting elements of secondary structure. The catalytic residues Cys-146, His-40, and Glu-71 are positioned as in serine proteases, but the oxyanion hole is moved 1-1.2 A away. Residues that bind to the 5' noncoding region of rhinovirus genomic RNA are located on the opposite side of the molecule from the active site. Interactions between individual 3Cpro molecules in the crystal lattice suggest a model for intermolecular proteolytic cleavage of the 3CD polyprotein.
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PMID:Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. 751 72

Molecular mechanics and dynamics combined with semiempirical calculations were carried out for purposes of comparison of the active site characteristics of AChE, trypsin, and chymotrypsin as probed by their diastereomeric adducts with 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman), methylphosphonate monoester anions, and tetravalent carbonyl intermediates of the reactions of the natural substrates in each case. Glu199 is a key residue in the electrostatic catalytic mechanism of AChE, in removal of the leaving group, and possibly by acting as an alternate general base catalyst. "Pushing" of an alkoxy ligand by Glu199 and the numerous small van der Waals interactions promote dealkylation in phosphonate adducts of AChE much more effectively than any other enzyme. A high concentration of negative charge created by the phosphonate ester monoanion and Glu199 adjacent to it fully accounts for the resistance to the attack of even the strongest nucleophile applied for enzyme reactivation. Stabilization of the developing negative charge on the phosphonates in the soman-inhibited PSCS adducts of serine hydrolases is by electrophilic residues in the oxyanion hole (AChE) and the protonated catalytic His. PR diastereomers of soman-inhibited AChE can be accommodated in an orientation in which the oxyanion hole interactions are lost and for which the stabilizing interactions are 17-26 kcal/mol smaller than in the PS diastereomer. The dealkylation reaction is almost equally likely in all diastereomers of soman-inhibited AChE. The stabilizing interaction energies are approximately 4 kcal/mol greater in the PR than in the PS adducts of the soman-inhibited serine proteases. There is 0.60 unit greater partial negative charge on the phosphonyl fragment in the anion of phosphonate monoesters of Ser than at the oxygens of tetravalent carbonyl transients resulting in approximately 12-22 kcal/mol greater stabilization of the former than the latter.
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PMID:Origins and diversity of the aging reaction in phosphonate adducts of serine hydrolase enzymes: what characteristics of the active site do they probe? 761 98

The inhibitory mechanism of trans-4-aminomethylcyclohexanecarbonyl-L-phenyl-alanine-4-carbo xymethylanilide (1), a noncovalent serine protease inhibitor synthesized based on previous structure-activity studies, was clarified based on the X-ray crystal structure of the complex (2.2 A resolution, R = 0.175), where the amino group of the aminomethylcyclohexane moiety was bifurcately hydrogen-bonded to the carboxyl oxygens of Asp 189 side group (specificity pocket), and the hydrogen bonds of the cyclohexanecarbonyl oxygen to NHs of Gly 193 and Ser 195 residues (oxyanion hole) and of Phe NH to Ser 195 O gamma atom (catalytic triad) were observed. In contrast, the Phe benzene moiety and terminal carboxymethylanilide of 1 were not well located on the electron density map, suggesting the conformational freedom of these P1' and P2' sites at the binding pocket. Based on these insights, trans-4-aminomethylcyclohexanecarbonyl-4-nitro-L-phenylalanine-4-+ ++benzoylanilide (2) was designed, in which the P1' and P2' sites were modified so as to effectively interact with the amino acid residues of trypsin binding pocket via hydrogen bonding and van der Waals interactions, respectively. Consequently, 2 showed 40 times higher inhibitory activity against trypsin than 1.
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PMID:Design of noncovalent trypsin inhibitor based on the X-ray crystal structure of the complex. 764 15

A mechanism of hemolytic hole formation during rapid hemolysis in a hypotonic medium has been investigated using eosin-5-maleimide (EMI) as a probe. The EMI-labeled erythrocytes revealed a distinct cluster and/or ring of intense fluorescence staining in a hypotonic 5 mM Hepes buffer (pH 7.4), but not in an isotonic buffer containing 150 mM KCl. This EMI cluster indicates an association of band 3 proteins, which correspond to a hemolytic hole. The hole was confirmed by an atomic force microscopy image. The erythrocytes showed a single large hole in the membrane. By the use of EMI-labeled ghosts, it was observed that the lateral clustering of band 3 was accompanied by a biphasic change of fluorescence intensity of EMI. This biphasic change is interpreted as the hemolytic hole formation by band 3, followed by a disappearance of the hole accompanied by band 3 diffusion or distribution within membrane. The latter event corresponds to a spontaneous membrane seal. When a cytoplasmic domain of band 3 was digested with trypsin, or when SH groups in the cytoplasm-facing components of the membrane were also labeled by EMI, no fluorescence change was observed. These results suggest that the association and/or dissociation of band 3 proteins in a hypotonic medium are strongly influenced by cytoplasmic domains. The apparent biphasic change of the fluorescence intensity in the hypotonic medium was well explained by assuming three events: swelling, clustering of band 3, and sealing accompanied by band 3 redistribution.
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PMID:Mechanism of hypotonic hemolysis of human erythrocytes. 768 91

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.
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PMID:Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects. 769 86

Trypsin is inactivated by the levorotatory enantiomers (most likely PS) of 4-nitrophenyl 4-H-, 4-CH3-,4-OCH3-, and 4-Cl-phenacyl methylphosphonates (PMNs) with second-order rate constants between 231 and 884 M-1 s-1. 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The second-order rate constants for the inactivation of alpha-chymotrypsin by the levorotatory enantiomers of the five PMNs are between 37,000 and 770,000 M-1 s-1, and those for the dextrorotatory enantiomers are between 400 and 640 M-1 s-1; the enantioselectivity is 90-1880. Specific rotation [alpha]22D of the faster-reacting enantiomer of 4-CH3-PMN with trypsin and alpha-chymotrypsin is -30 +/- 6 degrees. 31P NMR of the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the parent compound. Results of molecular mechanics and dynamics calculations show that the principal interactions are between the phosphonyl group and constituents of the oxyanion hole and between the aromatic fragment and residues in the binding regions of the enzymes. Trypsin activity returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the series. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the second to minute time scale. The data support a mechanism of reactivation involving rate-determining intramolecular displacement of Ser by the carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for trypsin reactivation from its adducts indicate involvement of an ionizable group with pKa approximately 8.0. The pH dependence and solvent isotope effects are small in most cases. The compounds demonstrate favorable properties for controllable and temporary modulation of enzyme activity.
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PMID:Enantioselective and reversible inhibition of trypsin and alpha-chymotrypsin by phosphonate esters. 802 18

The crystal structure of beta-lactamase from Staphylococcus aureus inactivated by p-nitrophenyl[[N-(benzyloxycarbonyl)amino]methyl]phosphonate, a methylphosphonate monoester monoanion inhibitor, has been determined and refined at 2.3 A resolution. The structure reveals a tetrahedral phosphorus covalently bonded to the O gamma atom of the active site serine, Ser70. One of the oxygen atoms bonded to phosphorus is located in the oxyanion hole formed by the two main-chain nitrogen atoms of Ser70 and Gln237, and the second bonded oxygen is solvated. The (benzyloxycarbonyl)aminomethyl group is oriented towards the active site gully such that the peptide group forms compensating electrostatic interactions with polar groups on the enzyme. The benzyl group forms a hydrophobic interaction with Ile239 and an aromatic-aromatic edge-to-face interaction with Tyr105, which has undergone a conformational transition relative to the native structure. The mode of binding supports the proposal that on reaction with the enzyme, the phosphonate generates a structure analogous to the tetrahedral transition state/intermediate associated with the acylation step of a normal substrate. The disposition of the phosphonyl group in this complex is the same as that of the corresponding phosphoryl group in the complex resulting from the inhibition of trypsin by diisopropylphosphofluoridate. The structure is consistent with a mechanism of inactivation that follows an associative pathway, proceeding via a transition state/intermediate in which phosphorus is penta-co-ordinated, forming a trigonal bipyramidal geometry with the phosphonyl donor (p-nitrophenol) and acceptor (Ser70 O gamma atom) in apical positions. A model of this transition state can be accommodated in the active site of beta-lactamase without any steric hindrance. A model of the tetrahedral transition state associated with the acylation step by benzyl penicillin has been derived. Because of the conformational rigidity of the fused rings of penicillin molecules, the orientation of the substrate is fixed once the tetrahedral carbonyl carbon and its ligands are superimposed on the phosphonate group. The outcome is that the carboxylate substituent on the thiazolidine ring forms a salt bridge with Lys234, and the preferred puckering of the ring is that observed in the crystal structure of ampicillin, the so-called "open" conformer.
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PMID:Structure of a phosphonate-inhibited beta-lactamase. An analog of the tetrahedral transition state/intermediate of beta-lactam hydrolysis. 823 Jan 96

Raman, absorbance, and kinetic measurements were used to determine how the serine protease active site feature known as the oxyanion hole interacts with an acyl-enzyme intermediate. The substrate, p-(dimethylamino)benzoylimidazolide (DAB-Im), was synthesized and used to prepare DAB-acyl-enzymes of wild-type (WT) and N155G subtilisin-BPN' (the N155G mutant lacks a fully functioning oxyanion hole), alpha-chymotrypsin (CHT), and bovine trypsin (TRY). DAB-acyl-enzyme deacylation rate constants, k3, were found to span a 720-fold range at pH 7.8 (DAB-WT > DAB-TRY > DAB-N155G > DAB-CHT). DAB-N155G was found to deacylate 80-fold slower than DAB-WT, indicating a 2.6 kcal/mol loss of transition-state binding energy due to this mutation. Absorbance spectra revealed strongly red-shifted absorbance lambda max values for all of the DAB-acyl-enzymes. The red shift was found to be 2.0 nm less in DAB-N155G, indicating that the oxyanion hole is partially responsible for this electronic perturbation of the DAB chromophore at the active site. Raman difference spectra of the DAB-acyl-enzymes measured at pH 5.0 and 8.6, with 18O-labeling of the carbonyl, show that the molecular motions most perturbed by the active site are three associated with the scissile acyl bond. Most interesting is the carbonyl stretching vibration, v(C = O), whose motion extends into the hydrolytic reaction coordinate. Comparison of the v(C = O) of DAB-WT and DAB-N155G reveals that the oxyanion hole does indeed form a hydrogen-bonding interaction with the carbonyl oxygen, the strength of which increases at pH 8.6. Interestingly, the DAB-TRY carbonyl forms very strong hydrogen bonds, even at pH 5.0, but DAB-CHT does not, even at pH 8.6. The low-frequency (1661 cm-1) v(C = O)'s of pH 5.0 DAB-TRY and pH 8.6 DAB-WT are proposed to correspond to a tetrahedrally distorted carbonyl center like that observed in the crystal structure of guanidinobenzoyl-TRY (Mangel et al., 1990). The strength of hydrogen bonding between the DAB-acyl-enzyme's carbonyl and the oxyanion hole, as gauged by the v(C = O) frequency, was found to correlate positively with an increased deacylation rate. This correlation, as well as calculated acyl-enzyme carbonyl bond lengths, which indicate a 0.015-A lengthening due to the oxyanion hole interaction, was found to be in good agreement with previously published resonance Raman data of alpha, beta-unsaturated arylacryloyl-acyl-enzymes (Tonge & Carey, 1990b, 1992).
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PMID:Details of the acyl-enzyme intermediate and the oxyanion hole in serine protease catalysis. 828 85

X-ray structures of trypsin from bovine pancreas inactivated by diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl)methanephosphonate [Z-(4-AmPhGly)P(OPh)2] were determined at 113 and 293 K to 1.8 angstrom resolution and refined to R factors of 0.211 (113 K) and 0. 178 (293 K). The structures reveal a tetrahedral phosphorus covalently bonded to the O gamma of the active site serine. Covalent bond formation is accompanied by the loss of both phenoxy groups. The D-stereoisomer of Z-(4-AmPhGly)P-(OPh)2 is not observed in the complex. The L-stereoisomer of the inhibitor forms contacts with several residues in the trypsin active site. One of the phosphonate oxygens is inserted into the oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp194, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N epsilon 2 of His 57. The p-amidinophenylglycine moiety binds into the trypsin primary specificity pocket, interacting with Asp189. The amide forms a hydrogen bond to the carbonyl oxygen atom of Ser214. The inhibitor moiety, from the 113 K structure of trypsin inactivated by the reaction product of Z-(4-AmPhGly)P(OPh)2, was docked into human thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., & Hofsteenge, J. (1989) EMBO J. 8, 3467-3475] and energy minimized. The inhibitor fits well into the thrombin active site, forming favorable contacts similar to those in the trypsin complex with no bad contacts.
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PMID:Inhibition of trypsin and thrombin by amino(4-amidinophenyl)methanephosphonate diphenyl ester derivatives: X-ray structures and molecular models. 860 48

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.
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PMID:Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. 860 1


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