Linked Life Data

10325403

Source:http://linkedlifedata.com/resource/pubmed/id/10325403

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
1999-7-16
pubmed:abstractText
We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme-substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4. 5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40 degrees C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0269-2139
pubmed:author
pubmed:issnType
Print
pubmed:volume
12
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
327-31
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation.
pubmed:affiliation
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't