Linked Life Data

14529297

Source:http://linkedlifedata.com/resource/pubmed/id/14529297

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
40
pubmed:dateCreated
2003-10-7
pubmed:abstractText
The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2'-deoxy-FAD. The k(cat) (18.8 +/- 0.5 s(-1)) and K(m) (2.5 +/- 0.4 microM) values for the oxidation of n-octanoyl-CoA (C(8)-CoA) by WT pMCAD recombinantly expressed in Escherichia coli are similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999) Biochemistry 38, 257-267], reconstitution of the WT enzyme with 2'-deoxy-FAD causes a large (400-fold) decrease in k(cat) but has little effect on K(m). To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2'-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm(-1) decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm(-1), the enone band is only shifted 10 cm(-1) upon binding HD-CoA to 2'-deoxy-FAD pMCAD. Thus, removal of the 2'-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol(-1), while the activation energy for the reaction is raised 15 kJ mol(-1). In addition, removal of the 2'-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol(-1). Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C(8)-CoA alpha-protons with solvent deuterium, the 2'-deoxy-FAD-reconstituted enzyme catalyzes alpha-proton exchange at a rate (k(exc)) of 0.085 s(-1), which is only 4-fold slower than k(exc) for WT pMCAD (0.35 s(-1)). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for alpha-proton exchange, in contrast to its role in substrate oxidation, or the value of k(exc) for WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
14
pubmed:volume
42
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
11846-56
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:14529297-Acyl Coenzyme A, pubmed-meshheading:14529297-Acyl-CoA Dehydrogenase, pubmed-meshheading:14529297-Animals, pubmed-meshheading:14529297-Binding Sites, pubmed-meshheading:14529297-Catalysis, pubmed-meshheading:14529297-Flavin-Adenine Dinucleotide, pubmed-meshheading:14529297-Glutamic Acid, pubmed-meshheading:14529297-Glutamine, pubmed-meshheading:14529297-Hydrogen Bonding, pubmed-meshheading:14529297-Kinetics, pubmed-meshheading:14529297-Mutagenesis, Site-Directed, pubmed-meshheading:14529297-Potentiometry, pubmed-meshheading:14529297-Proline, pubmed-meshheading:14529297-Protons, pubmed-meshheading:14529297-Recombinant Proteins, pubmed-meshheading:14529297-Spectrophotometry, Ultraviolet, pubmed-meshheading:14529297-Spectrum Analysis, Raman, pubmed-meshheading:14529297-Swine
pubmed:year
2003
pubmed:articleTitle
Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy.
pubmed:affiliation
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't