Linked Life Data

19635449

Source:http://linkedlifedata.com/resource/pubmed/id/19635449

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2009-9-14
pubmed:abstractText
The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA-CoA thioesterase with up to four natural substrate (4-HBA-CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/or products in the millisecond time range.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1096-0309
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
394
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
209-16
pubmed:dateRevised
2010-12-3
pubmed:meshHeading
pubmed-meshheading:19635449-Arthrobacter, pubmed-meshheading:19635449-Binding Sites, pubmed-meshheading:19635449-Catalysis, pubmed-meshheading:19635449-Coenzyme A, pubmed-meshheading:19635449-Hydrogen-Ion Concentration, pubmed-meshheading:19635449-Kinetics, pubmed-meshheading:19635449-Ligands, pubmed-meshheading:19635449-Mass Spectrometry, pubmed-meshheading:19635449-Models, Chemical, pubmed-meshheading:19635449-Models, Molecular, pubmed-meshheading:19635449-Molecular Weight, pubmed-meshheading:19635449-Protein Binding, pubmed-meshheading:19635449-Protein Conformation, pubmed-meshheading:19635449-Protein Folding, pubmed-meshheading:19635449-Protein Structure, Quaternary, pubmed-meshheading:19635449-Spectrometry, Mass, Electrospray Ionization, pubmed-meshheading:19635449-Substrate Specificity, pubmed-meshheading:19635449-Thiolester Hydrolases
pubmed:year
2009
pubmed:articleTitle
Monitoring enzyme catalysis in the multimeric state: direct observation of Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry.
pubmed:affiliation
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural