Linked Life Data

15078103

Source:http://linkedlifedata.com/resource/pubmed/id/15078103

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
15
pubmed:dateCreated
2004-4-13
pubmed:abstractText
SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be critical for catalysis by mutating to Ala, while the Cys145 to Ser mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pK(a)'s for His41 and Cys145 in the wild-type enzyme were estimated to be 6.38 and 8.34, while the pK(a)'s for His41 and Ser145 in the C145S mutant were estimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D(2)O for general base catalysis, that is, reacts slower in D(2)O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pK(a) values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
43
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
4568-74
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:15078103-Alanine, pubmed-meshheading:15078103-Amino Acid Substitution, pubmed-meshheading:15078103-Catalysis, pubmed-meshheading:15078103-Colorimetry, pubmed-meshheading:15078103-Cysteine, pubmed-meshheading:15078103-Cysteine Endopeptidases, pubmed-meshheading:15078103-Deuterium Exchange Measurement, pubmed-meshheading:15078103-Endopeptidases, pubmed-meshheading:15078103-Histidine, pubmed-meshheading:15078103-Hydrogen-Ion Concentration, pubmed-meshheading:15078103-Hydrolysis, pubmed-meshheading:15078103-Kinetics, pubmed-meshheading:15078103-Mutagenesis, Site-Directed, pubmed-meshheading:15078103-SARS Virus, pubmed-meshheading:15078103-Serine, pubmed-meshheading:15078103-Solvents, pubmed-meshheading:15078103-Substrate Specificity, pubmed-meshheading:15078103-Temperature, pubmed-meshheading:15078103-Thermodynamics, pubmed-meshheading:15078103-Viral Proteins
pubmed:year
2004
pubmed:articleTitle
3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.
pubmed:affiliation
State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't