Linked Life Data

6690998

Source:http://linkedlifedata.com/resource/pubmed/id/6690998

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5947
pubmed:dateCreated
1984-2-23
pubmed:abstractText
A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme-substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 leads to Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 leads to Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
0028-0836
pubmed:author
pubmed:issnType
Print
pubmed:volume
307
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
187-8
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:articleTitle
A large increase in enzyme-substrate affinity by protein engineering.
pubmed:publicationType
Journal Article