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

---

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

Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site".
...
PMID:Human alpha-thrombin inhibition by the active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: a comparative kinetic and X-ray crystallographic study. 863 15

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.
...
PMID:Two crystal structures of the leupeptin-trypsin complex. 884 65

Factor D (D) is a serine protease essential in the activation of the alternative complement pathway. Only a few of the common serine protease inhibitors inhibit D, binding covalently to the serine hydroxyl of the catalytic triad. 3,4-Dichloroisocoumarin (DCI) is a mechanism-based inhibitor which inhibits most serine proteases and many esterases, including D. The structure of the enzyme:inhibitor covalent adduct of D with DCI, DCI:D, to a resolution of 1.8 A is described, which represents the first structural analysis of D with a mechanism-based inhibitor. The side chain of the ring-opened DCI moiety of the protein adduct undergoes chemical modification in the buffered solution, resulting in the formation of an alpha-hydroxy acid moiety through the nucleophilic substitution of both Cl atoms. The inhibited enzyme is similar in overall structure to the native enzyme, as well as to a variety of isocoumarin-inhibited trypsin and porcine pancreatic elastase (PPE) structures, yet notable differences are observed in the active site and binding mode of these small-molecule inhibitors. One region of the active site (residues 189-195) is relatively conserved between factor D, trypsin, and elastase with respect to amino-acid sequence and to conformation. Another region (residues 214-220) reflects the amino-acid substitutions and conformational flexibility between these enzymes. The carbonyl O atom of the DCI moiety was found to be oriented away from the oxyanion hole, which greatly contributes to the stability of the DCI:D adduct. The comparisons of the active sites between native factor D, DCI-inhibited factor D, and various inhibited trypsin and elastase (PPE) molecules are providing the chemical bases directing our design of novel, small-molecule pharmaceutical agents capable of modulating the alternative complement pathway.
...
PMID:Structure of 3,4-dichloroisocoumarin-inhibited factor D. 975 85

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].
...
PMID:Oxyanion-mediated inhibition of serine proteases. 983 2

A matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer was developed which uses a novel reflectron composed of a grounded cylinder and an adjustable endcap electrode to provide high-order kinetic energy focusing for a miniaturized mass analyzer. The nearly quadratic potential form of the reflecting field focuses ions desorbed from a source of very small dimensions formed by placing the sample probe within the centered hole of the coaxial dual channel plate detector. At the same time, the depth of the reflectron can be adjusted to accommodate a short drift length between the source/detector and the reflectron. For larger drift lengths, in particular to allow the addition of an XY stage for the analysis of sample arrays, endcap reflectron focusing can be combined with time-delayed ion extraction to achieve good mass resolution. The instrument has been used for the analysis of peptides digested with trypsin or carboxypeptidase, and also small DNA oligomers.
...
PMID:Miniaturized time-of-flight mass spectrometer for peptide and oligonucleotide analysis. 1058 34

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment.
...
PMID:Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution. 1073 44

We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin. Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.
...
PMID:A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site. 1129 54

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.
...
PMID:The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity. 1142 Apr 35

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lactoferrin fragment.
...
PMID:Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution. 1156 28

The contribution of electrostriction of the solvent to the stabilization of the negatively charged tetrahedral transition state of a trypsin-catalyzed reaction was probed by means of kinetic studies involving high-pressure and solvent dielectric constant. A good correlation was observed between the increased catalytic efficiency of trypsin and the decreased solvent dielectric constant. When the dielectric constant of the solvents was lowered by 4.68 units, the loss of activation energy and that of free energy of activation were 2.26 kJ/mol and 3.09 kJ/mol, respectively. The activation volume for k(cat) decreased significantly as the dielectric constant of the solvent decreased, indicating that the degree of electrostriction of the solvent around the charged tetrahedral transition state has been enhanced. These observations demonstrate that the increase in the catalytic efficiency of the trypsin reaction with decreasing dielectric constant resulted from the stabilization of electrostatic energy for the formation of an oxyanion hole, and this stabilization was caused by the increase of electrostricted water around the charged tetrahedral transition state. Therefore, we conclude that control of the solvent dielectric constant can stabilize the tetrahedral transition state, and this lowers the activation energy.
...
PMID:The enhancement of electrostriction caused by lowering the solvent dielectric constant leads to the decrease of activation energy in trypsin catalysis. 1173 Oct 85


<< Previous 1 2 3 4 5 6 Next >>

---