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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
Pt 2
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pubmed:dateCreated |
2005-7-4
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pubmed:abstractText |
Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo-keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80-->Ala (K80A) and Asp-46-->Asn Lys-80-->Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd = 24 microM) and NADP+ (Kd = 0.03 microM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd = 51 microM) and NADPH (Kd = 19 microM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M(-1).s(-1) for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope = +0.29; r2 = 0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (+/-0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-10418995,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-10610783,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-10661866,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-10681056,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-11123955,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-11445075,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-11513616,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-11828433,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-11847285,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12003638,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12102621,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12604248,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12733986,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12826012,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12855766,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-12962477,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-15095000,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-15109252,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-15544321,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-15581900,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-1621098,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-2674899,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-7578039,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-7669785,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-7742302,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-8117659,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-8245005,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-9307009,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-9521675,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-9600897,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-9657682,
http://linkedlifedata.com/resource/pubmed/commentcorrection/15799715-9765214
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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 |
Jul
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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 |
15
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pubmed:volume |
389
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
507-15
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:15799715-Aldehyde Reductase,
pubmed-meshheading:15799715-Binding Sites,
pubmed-meshheading:15799715-Candida,
pubmed-meshheading:15799715-Catalysis,
pubmed-meshheading:15799715-Gene Expression Regulation, Fungal,
pubmed-meshheading:15799715-Genetic Complementation Test,
pubmed-meshheading:15799715-Hydrogen-Ion Concentration,
pubmed-meshheading:15799715-Kinetics,
pubmed-meshheading:15799715-Mutagenesis, Site-Directed,
pubmed-meshheading:15799715-Mutation,
pubmed-meshheading:15799715-NADP,
pubmed-meshheading:15799715-Oxidation-Reduction,
pubmed-meshheading:15799715-Pargyline,
pubmed-meshheading:15799715-Propylamines,
pubmed-meshheading:15799715-Protein Binding,
pubmed-meshheading:15799715-Static Electricity,
pubmed-meshheading:15799715-Substrate Specificity,
pubmed-meshheading:15799715-Xylitol,
pubmed-meshheading:15799715-Xylose
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pubmed:year |
2005
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pubmed:articleTitle |
Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies.
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pubmed:affiliation |
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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