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)

Two closely related kallikrein-like proteinases having little activity toward the standard synthetic amide substrates of tissue kallikreins were isolated from the rat submandibular gland. They were found to be the protein products of the rKlk2 (tonin) and the rKlk9 genes by amino acid sequence analysis (nomenclature of the genes and proteins of the kallikrein family is according to the proposal of the discussion panel from the participants of the KININ '91 meeting held Sept. 8-14, 1991, in Munich, Germany). These two proteinases of similar structure also had very similar physicochemical properties. They differed from other kallikrein-related proteinases in having high pHi values of 6.20 (rK2) and 6.85 (rK9). Kallikrein rK2 was purified as a single peptide chain, whereas rK9 appeared as a two-chain protein after reduction. Their enzymatic properties were also very similar and differed significantly from those of other rat kallikrein-related proteinases. Unlike the five other kallikrein-related proteinases we have purified so far, kallikrein rK9 was not inhibited by aprotinin. rK9 also differed from rK2 by its tissue localization. The prostate gland contained only rK9 where it was the major kallikrein-like component. The amino acids preferentially accommodated by the proteinase S3 to S2' subsites were identified using synthetic amide and protein substrates. Unlike other kallikrein-related proteinases, rK2 had a prevalent chymotrypsin-like specificity, whereas rK9 had both chymotrypsin-like and trypsin-like properties. Both rK2 and rK9 preferred a prolyl residue in position P2 of the substrate and did not accommodate bulky and hydrophobic residues at that position, as did most of the other kallikrein-related proteinases. This P2-proline-directed specificity is necessary for processing the precursors of several biologically active peptides. Subsites accommodating residues COOH-terminal to the scissile bond were also important in determining the overall substrate specificity of these proteinases. rK2 and rK9 both showed a preference for hydrophobic residues in P2'. Other subsites upstream of the S3 subsite were found to intervene in substrate binding and hydrolysis. The restricted specificity of rK2 and rK9 is consistent with the presence of an extended substrate binding site, and hence with a processing enzyme function. Their P1 specificities enabled both proteinases to release angiotensin II from angiotensinogen and from angiotensinogen I, but rK9 was at least 100 times less active than rK2 on both substrates. The substrate specificities of rK2 and rK9 were correlated with key amino acids defining their substrate binding site.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Protein products of the rat kallikrein gene family. Substrate specificities of kallikrein rK2 (tonin) and kallikrein rK9. 131 52

An easy and rapid enzymatic method is described which allows replacement of P'-residues in bovine pancreatic trypsin inhibitor. Insertion of Xaa-Arg or Xaa-Lys into a BPTI fragment lacking P1' = Ala16 and P2' = Arg17 was carried out in a "one pot" reaction catalysed by trypsin in the presence of 80% 1,4 butanediol.
...
PMID:Enzymatic fragment substitution as a tool in protein design. 172 55

Site-specific mutagenesis techniques have been used to construct active site variants of the Kunitz-type protease inhibitor domain present in the Alzheimer's beta-amyloid precursor protein (APP-KD). Striking alteration of its protease inhibitory properties were obtained when the putative P1 residue, arginine, was replaced with the small hydrophobic residue valine. The altered protein was no longer inhibitory toward bovine pancreatic trypsin, human Factor XIa, mouse epidermal growth factor-binding protein, or bovine chymotrypsin, all of which are strongly inhibited by the unaltered APP-KD (Sinha, S., Dovey, H. F., Seubert, P., Ward, P. J., Blacher, R. W., Blaber, M., Bradshaw, R. A., Arici, M., Mobley, W. C., and Lieberburg, I. (1990) J. Biol. Chem. 265, 8983-8985). Instead, the P1-Val-APP-KD was a potent inhibitor of human neutrophil elastase, with a Ki = 0.8 nM, as estimated by the inhibition of the activity of human neutrophil elastase measured using a chromogenic substrate. It also inhibited the degradation of insoluble elastin by the enzyme virtually stoichiometrically. Replacement of the P1' (Ala) and P2' (Met) residues of P1-Val-MKD with the corresponding residues (Ser, Ile) from alpha 1-proteinase inhibitor resulted in an inactive protein, underscoring the mechanistic differences between the serpins from the Kunitz-type protease inhibitor family. These results confirm the importance of the P1 arginine residue of APP-KD in determining inhibitory specificity, and are also the first time that a single amino acid replacement has been shown to generate a specific potent human neutrophil elastase inhibitor from a human KD sequence.
...
PMID:Conversion of the Alzheimer's beta-amyloid precursor protein (APP) Kunitz domain into a potent human neutrophil elastase inhibitor. 193 50

A series of acetyl-peptidyl-amides containing the amino acid sequence around the Arg-Ser kallikrein cleavage site of bovine kininogen were synthesized and tested for their ability to inhibit both the kinin-releasing activity and the amidase activity of purified human urinary kallikrein. The substrate analogues were competitive inhibitors for human urinary kallikrein and the heptapeptides (P4-P3'), hexapeptides (P3-P3'), and pentapeptides (P2-P3') gave Ki values of 140, 64, and 18 microM respectively, while the tetrapeptides (P1-P3'), tripeptides (P1'-P3') and dipeptides (P2'-P3') had little or no inhibitory activity. The effective analogues had neither kinin-like nor kinin-blocking activity on the rat uterus either before or after exposure to human urinary kallikrein. The effective human urinary kallikrein inhibitors were further examined for their effect on other serine proteases, including human plasma kallikrein, plasmin, complement components (C1s, C1r), bovine coagulation factors (IIa, IXa, and Xa), elastase, and trypsin. These peptides showed little inhibition of the circulating serine proteases but yielded a Ki for the nonspecific protease trypsin in the microM range. These results should provide the basis for the development of highly specific tissue kallikrein inhibitors to aid in elucidating the in vivo role(s) of tissue kallikreins.
...
PMID:Specificity of substrate analogue inhibitors of human urinary kallikrein. 384 67

In an effort to further develop the technique of isomer-specific proteolysis, a number of proline-containing substrates were subjected to hydrolysis in the presence of chymotrypsin, trypsin, or prolidase. The objective was to determine whether direct hydrolysis of the cis form of the substrate could occur and, if so, the extent to which it is slower than the hydrolysis of the equivalent trans form. It is shown that for both peptide and amide substrates, which contain proline at the P2 position, the cis form can be hydrolyzed directly by either chymotrypsin or trypsin, in contrast to earlier suggestions in the literature. For similar amide substrates, it was found that chymotrypsin has a lower catalytic efficiency for the cis form, relative to the trans form, by a factor of 20 000 while, for trypsin and its substrate, the cis form was cleaved about 2000 times less efficiently. Results for a trypsin substrate with proline at the P2' position, rather than the P2 position, were quite different however, since there was no indication that the cis form could be directly cleaved even at the highest enzyme concentration. There was also no indication that prolidase could cleave the dipeptide Phe-Pro when the active bond itself is in the cis form. These collective results suggest that the ability of proteases to cleave a substrate with a cis peptide bond depends strongly on the location of the cis bond relative to the active bond that is being cleaved.
...
PMID:Isomer-specific proteolysis of model substrates: influence that the location of the proline residue exerts on cis/trans specificity. 408 35

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.
...
PMID:Design of noncovalent trypsin inhibitor based on the X-ray crystal structure of the complex. 764 15

Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme-substrate model suggested that histidine specificity at the P2' position might be achieved by a tridentate site involving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'-His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfavored by trypsin, this result demonstrates that a metal cofactor can be used to modulate specificity in a designed fashion. The same geometric criteria applied in the primary S1 binding pocket suggested that the single-site mutation D189H might effect metal-dependent His specificity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His189 rotates away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modification at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate P1 side chains. By taking advantage of the extended subsites, an alternate substrate specificity has been engineered into trypsin.
...
PMID:Engineered metal regulation of trypsin specificity. 785 28

Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189S exhibits a 25-fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc. With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites.
...
PMID:Delocalizing trypsin specificity with metal activation. 863 40

The three-dimensional structures of complexes of trypsin N143H, E151H bound to ecotin A86H are determined at 2.0 A resolution via X-ray crystallography in the absence and presence of the transition metals Zn2+, Ni2+, and Cu2+. The binding site for these transition metals was constructed by substitution of key amino acids with histidine at the trypsin-ecotin interface in the S2'/P2' pocket. Three histidine side chains, two on trypsin at positions 143 and 151 and one on ecotin at position 86, anchor the metals and provide extended catalytic recognition for substrates with His in the P2' pocket. Comparisons of the three-dimensional structures show the different geometries that result upon the binding of metal in the engineered tridentate site and suggest a structural basis for the kinetics of the metal-regulated catalysis. Of the three metals, the binding of zinc results in the most favorable binding geometry, not dissimilar to those observed in naturally occurring zinc binding proteins.
...
PMID:X-ray structures of a designed binding site in trypsin show metal-dependent geometry. 863 41

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the relationship of their reactive-loop conformations to those of circulating forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and anti-thrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop peptides. Proteolysis at the P10-P9, P8-P7 and P7-P6 of anti-trypsin was distorted by binary complex formation. The P1'-P2' bond in anti-thrombin was more accessible to proteolysis after binary complex formation, whereas cleavage at the P4-P3 bond was variably altered by synthetic peptide insertion. The proteolytic accessibility of the reactive-site P1-P1' bond of anti-trypsin and anti-thrombin binary complexes was identical with that of the native form and no cleavage was observed in the hinge region (P15-P10) of either protein, whether native or as binary complexes. these results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in the circulation, with the hinge region being partly incorporated into the A beta-pleated sheet. This loop can be displaced by peptides and induced to adopt a new conformation similar to the three-turn helix of ovalbumin. Both the native and binary complexed forms of anti-thrombin showed a greatly increased proteolytic sensitivity in the presence of heparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates the approximation of enzyme and inhibitor.
...
PMID:Probing serpin reactive-loop conformations by proteolytic cleavage. 867 81


1 2 3 4 Next >>

---