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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
Pt 1
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pubmed:dateCreated |
2003-9-22
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pubmed:abstractText |
X-ray structure of the Pseudomonas fluorescens mannitol 2-dehydrogenase ternary complex with NAD+ and D-mannitol suggests that Lys-295 provides catalytic base assistance to secondary alcohol group oxidation. We have replaced Lys-295 by site-directed mutagenesis with alanine or methionine and evaluated the catalytic significance of side-chain substitution by kinetic analysis of restoration of activity with external amines, and from pH and solvent isotope effects on the reaction catalysed by K295A (Lys-295-->Ala mutant). K295A and K295M (Lys-295-->Met mutants) show 3x10(4)- and 2x10(6)-fold lower turnover numbers respectively for D-mannitol oxidation (kcatO) at pH 10.0 than the wild-type. The second-order rate constant for non-covalent rescue of activity (kB) by free methylamine base is 31 M(-1) x s(-1) for K295A, but only 0.021 M(-1) x s(-1) for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope beta=0.66+/-0.16; r2=0.99) for K295A-catalysed D-mannitol oxidation at pH 10.0. The kcatO values of K295A in H2O and 2H2O are linearly dependent on [OL-] in the pL range 7.5-10.5 (where L is 1H or 2H). The solvent isotope effect on kcatO is 0.69. The time course of D-fructose reduction by K295A at pH 8.2 displays a pre-steady-state burst of NADH consumption. These data support a mechanism in which the epsilon -NH2 group of Lys-295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of enzyme-bound D-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-10441145,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-10471272,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-10841783,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-10864505,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-11123955,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-11724562,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-12146981,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-12175334,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-12196534,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-12604241,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-12604242,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-2161681,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-6294457,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-6395897,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-6688159,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-6987061,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-7013801,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-7016531,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-7922042,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-9730832,
http://linkedlifedata.com/resource/pubmed/commentcorrection/12826012-9843374
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Oct
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pubmed:issn |
1470-8728
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:day |
1
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pubmed:volume |
375
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
141-9
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:12826012-Alcohol Oxidoreductases,
pubmed-meshheading:12826012-Amines,
pubmed-meshheading:12826012-Catalysis,
pubmed-meshheading:12826012-Deuterium,
pubmed-meshheading:12826012-Hydrogen-Ion Concentration,
pubmed-meshheading:12826012-Kinetics,
pubmed-meshheading:12826012-Lysine,
pubmed-meshheading:12826012-Mannitol,
pubmed-meshheading:12826012-Mannitol Dehydrogenases,
pubmed-meshheading:12826012-Models, Chemical,
pubmed-meshheading:12826012-Mutation,
pubmed-meshheading:12826012-Oxidation-Reduction,
pubmed-meshheading:12826012-Pseudomonas fluorescens,
pubmed-meshheading:12826012-Solvents
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pubmed:year |
2003
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pubmed:articleTitle |
On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.
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pubmed:affiliation |
Institute of Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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