Linked Life Data

19857201

Source:http://linkedlifedata.com/resource/pubmed/id/19857201

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2009-12-22
pubmed:abstractText
The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1470-8728
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
425
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
455-63
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites.
pubmed:affiliation
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
pubmed:publicationType
Journal Article