Linked Life Data

19053262

Source:http://linkedlifedata.com/resource/pubmed/id/19053262

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
52
pubmed:dateCreated
2008-12-23
pubmed:databankReference
pubmed:abstractText
The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions. This is relevant for the genetic engineering of crops with economic interest that do not naturally possess efficient pathways for the synthesis of glycine betaine and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, the best characterized choline-oxidizing enzyme is the flavin-dependent choline oxidase from Arthrobacter globiformis, for which structural, mechanistic, and biochemical data are available. Here, we have replaced a hydrophobic residue (Val464) lining the active site cavity close to the N(5) atom of the flavin with threonine or alanine to investigate its role in the reaction of choline oxidation catalyzed by choline oxidase. The reductive half-reactions of the enzyme variants containing Thr464 or Ala464 were investigated using substrate and solvent kinetic isotope effects, solvent viscosity effects, and proton inventories. Replacement of Val464 with threonine or alanine uncovered a kinetically slow equilibrium between a catalytically incompetent form of enzyme and an active species that can efficiently oxidize choline. In both variants, the active form of enzyme shows a decreased rate of hydroxyl proton abstraction from the alcohol substrate, with minimal changes in the subsequent rate of hydride ion transfer to the flavin. This study therefore establishes that a hydrophobic residue not directly participating in catalysis plays important roles in the reaction of choline oxidation catalyzed by choline oxidase.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1520-4995
pubmed:author
pubmed:issnType
Electronic
pubmed:day
30
pubmed:volume
47
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
13850-61
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Substitution of an active site valine uncovers a kinetically slow equilibrium between competent and incompetent forms of choline oxidase.
pubmed:affiliation
Departments of Chemistry and Biology, The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't