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 primary processing of the poliovirus polyprotein is catalyzed by 2A protease (2Apro) which cleaves at the 1D/2A junction in a very rapid cotranslational reaction. In addition, 2Apro also indirectly induces cleavage of the p220 component of eIF-4F, which results in selective inhibition of host protein synthesis. Earlier studies have indicated that 2Apro is related to 3C protease (3Cpro) and is structurally similar to trypsin-like serine proteases with the substitution of Cys109 as the nucleophile. We noticed that 2Apro of enteroviruses and rhinoviruses contains a specific motif of Cys55-Xaa-Cys57-Xaan-Cys115-Xaa-His117 which is absolutely conserved, but which is not found in viral 3Cpro or known cellular serine proteases. To better understand the specific roles these conserved cysteine and histidine residues played in the structure/function of 2Apro, we constructed a series of 2Apro mutants by site-specific mutagenesis and analyzed the mutant enzymes with respect to their biochemical properties. Conservative amino acid replacements at Cys55, Cys57, Cys115, or His117 resulted, in each case, in a complete loss of both in cis and in trans activities of 2Apro. To determine the function of these residues, we examined the biochemical/structural features of 2Apro expressed in a cell-free rabbit reticulocyte lysate system. Gel mobility shift and chemical modification data suggest that these cysteine residues do not form intra-molecular disulfide linkages as a structural feature of 2Apro. However, studies with metal chelators did not eliminate the possibility that 2Apro contains a metal-binding ligand. Finally, our results suggest that these conserved cysteine and histidine residues, including Cys55, Cys57, Cys115, and His117, are critical in maintaining the active conformation of 2Apro structure and essential in supporting the catalytic activity of 2Apro.
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PMID:Characterization of the roles of conserved cysteine and histidine residues in poliovirus 2A protease. 131 Jan 93

The picornavirus 3C proteinases are substrate-specific thiol proteases that have been shown by secondary structure predictions and protein modeling studies to be similar to the trypsin-like serine proteases. We have examined several mutations of the 3C proteinase at putative active site and non-active site residues. The effect on 3C-mediated protein processing supports the model of serine protease similarity. In particular, we have shown that 3C can utilize a serine at position 147, which is predicted to supply the nucleophilic residue of the catalytic triad. We suggest that picornavirus 3C proteinases may represent a class of enzymes that have maintained the catalytic mechanism characteristic of a proposed enzyme ancestral to the highly divergent class of serine proteases.
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PMID:Poliovirus thiol proteinase 3C can utilize a serine nucleophile within the putative catalytic triad. 165 4

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.
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PMID:Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases. 215 90

To identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates. Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, involved in substrate binding, or present in nonconserved regions. Wild-type and mutant 3C proteins were expressed in Escherichia coli and tested for their ability to cleave synthetic substrates corresponding to different portions of the viral genome. Substitutions at His-46 (catalytic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) produced enzymes unable to process P1, P2, or P3 substrates in trans, whereas a change in the conserved Asp-98 had no effect on enzyme activity. Substitution of Ser for Cys-163 (catalytic triad) yielded an enzyme that retained activity on some substrates, while a substitution of Gly at this position resulted in a completely inactive enzyme. The kinetics of trans processing of translation products from a transcript encoding the P1 and P2 coding regions and the 2C/3A cleavage site with wild-type 3C or a transcript encoding P1 with 3C mutants revealed that the order of cleavage was VP3-VP1, VP0-VP3, VP1-2A, 2C-3A, and 2B-2C. Mutations in 3C that resulted in a partially active enzyme were individually introduced into full-length FMDV cDNA and RNA transcripts were translated in a cell-free system and used to transfect cells. In all cases the virus that was rescued had reverted to the wild-type 3C codon.
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PMID:Identification of the active-site residues of the 3C proteinase of foot-and-mouth disease virus. 749 82

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

Picornavirus 3C proteases are substrate-specific cysteine proteases, proposed to be homologous to the trypsin/chymotrypsin-like serine proteases on the basis of structural predictions. Substitutions at the putative active-site residues (Glu71 and Cys147) of the poliovirus 3C protease did not completely abolish proteolytic processing in vitro. The activity of mutated 3C proteases was in the following hierarchy: Glu71-Cys147 (wild type) > Asp71-Cys147 > Glu71-Ser147 > Gln71-Cys147 > Asp71-Ser147 > Gln71-Ser147 (inactive at all sites). Such mutations had differential effects on cleavage at different sites of the poliovirus polyprotein. Cleavage within the P1 region of the polyprotein was the most defective, at the 1ABC/VP1 junction and particularly at the VP0/VP3 junction. Cleavage at the 3AB/3CD and 2B/2C junctions was less affected by the mutations, and the P2/P3 and 2A/2BC junctions were cleaved efficiently by all mutants except Gln71-Ser147. All the 3C mutants gave negative results in infectivity and replication assays after transfection, indicating that mutation of Glu71 or Cys147 virtually abolishes viral replication, irrespective of the efficiency of processing of the nonstructural part of the polyprotein.
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PMID:Substitution mutations at the putative catalytic triad of the poliovirus 3C protease have differential effects on cleavage at different sites. 809 6

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.
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PMID:Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements. 966 69

The picornavirus family contains several human pathogens including human rhinovirus (HRV) and hepatitis A virus (HAV). In the case of HRVs, these small single-stranded positive-sense RNA viruses translate their genetic information into a polyprotein precursor which is further processed mainly by two viral proteases designated 2A and 3C. The 2A protease (2Apro) makes the first cleavage between the structural and non-structural proteins, while 3C protease (3Cpro) catalyzes most of the remaining internal cleavages. It has been shown that both 2Apro and 3Cpro are cysteine proteases but their overall protein folding is more like trypsin-type serine proteases. Due to their unique protein structure and essential roles in viral replication, 2Apro and 3Cpro have been viewed as excellent targets for antiviral intervention. In recent years, considerable efforts have been made in the development of antiviral compounds targeting these proteases. This article summarizes the recent approaches in the design of novel 2A and 3C protease inhibitors as potential antiviral agents for the treatment of picornaviral infections.
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PMID:Protease inhibitors as potential antiviral agents for the treatment of picornaviral infections. 1039 29

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.
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PMID:Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes. 1050 Jan 14

Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.
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PMID:Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of picornain 3C. 1152 3


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