Linked Life Data

12096909

Source:http://linkedlifedata.com/resource/pubmed/id/12096909

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2002-7-4
pubmed:abstractText
Understanding enzymes quantitatively and mimicking their remarkable catalytic efficiency is a paramount challenge. Here, we applied esterolytic antibodies (the D-Abs) to dissect and quantify individual elements of enzymatic catalysis such as transition state (TS) stabilization, nucleophilic reactivity and conformational changes. Kinetic and mutagenic analysis of the D-Abs were combined with existing structural evidence to show that catalysis by the D-Abs is driven primarily by stabilization of the tetrahedral oxyanionic intermediate of ester hydrolysis formed by the nucleophilic attack of an exogenous (solution) hydroxide anion. The side-chain of TyrH100d is shown to be the main H-bond donor of the D-Abs oxyanion hole. The pH-rate and pH-binding profiles indicate that the strength of this H-bond increases dramatically as the neutral substrate develops into the oxyanionic TS, resulting in TS stabilization of 5-7 kcal/mol, which is comparable to oxyanionic TS stabilization in serine hydrolases. We show that the rate of the exogenous (intermolecular) nucleophilic attack can be enhanced by 2000-fold by replacing the hydroxide nucleophile with peroxide, an alpha-nucleophile that is much more reactive than hydroxide. In the presence of peroxide, the rate saturates (k(cat)(max)) at 6 s(-1). This rate-ceiling appears to be dictated by the rate of the induced-fit conformational rearrangement leading to the active antibody-TS complex. The selective usage of negatively charged exogenous nucleophiles by the D-Abs led to the identification of a positively charged channel. Imprinted by the negatively-charged TS-analogue against which these antibodies were elicited, this channel presumably directs the nucleophile to the antibody-bound substrate. Our findings are discussed in comparison with serine esterases and, in particular, with cocaine esterase (cocE), which possesses a tyrosine based oxyanion hole.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0022-2836
pubmed:author
pubmed:copyrightInfo
(c) 2002 Elsevier Science Ltd.
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
320
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
559-72
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Esterolytic antibodies as mechanistic and structural models of hydrolases-a quantitative analysis.
pubmed:affiliation
Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.
pubmed:publicationType
Journal Article, Comparative Study, In Vitro, Research Support, Non-U.S. Gov't