Source:http://linkedlifedata.com/resource/pubmed/id/12795616
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
23
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| pubmed:dateCreated |
2003-6-10
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| pubmed:abstractText |
The chemical mechanism of a retaining beta-mannosidase from Cellulomonas fimi has been characterized through steady-state kinetic analyses with a range of substrates, coupled with chemical rescue studies on both the wild-type enzyme and mutants in which active site carboxyl groups have been replaced. Studies with a series of aryl beta-mannosides of vastly different reactivities (pK(a)(lg) = 4-10) allowed kinetic isolation of the glycosylation and deglycosylation steps. Substrate inhibition was observed for all but the least reactive of these substrates. Brønsted analysis of k(cat) revealed a downward breaking plot (beta(lg) = -0.54 +/- 0.05) that is consistent with a change in rate-determining step (glycosylation to deglycosylation), and this was confirmed by partitioning studies with ethylene glycol. The pH dependence of k(cat)/K(m) follows an apparent single ionization of a group of pK(a) = 7.65 that must be protonated for catalysis. The tentative assignment of E429 as the acid-base catalyst of Man2A on the basis of sequence alignments with other family 2 glycosidases was confirmed by the increased turnover rate observed for the mutant E429A in the presence of azide and fluoride, leading to the production of beta-mannosyl azide and beta-mannosyl fluoride, respectively. A pH-dependent chemical rescue of E429A activity is also observed with citrate. Substantial oxocarbenium ion character at the transition state was demonstrated by the alpha-deuterium kinetic isotope effect for Man2A E429A of alpha-D(V) = 1.12 +/- 0.01. Surprisingly, this isotope effect was substantially greater in the presence of azide (alpha-D(V) = 1.166 +/- 0.009). Likely involvement of acid/base catalysis was revealed by the pH dependence of k(cat) for Man2A E429A, which follows a bell-shaped profile described by pK(a) values of 6.1 and 8.4, substantially different from that of the wild-type enzyme. The glycosidic bond cleaving activity of Man2A E519A and E519S nucleophile mutants is restored with azide and fluoride and appears to correlate with the corresponding "glycosynthase" activities. The contribution of the substrate 2-hydroxyl to stabilization of the Man2A glycosylation transition state (DeltaDeltaG() = 5.1 kcal mol(-1)) was probed using a 2-deoxymannose substrate. This value, surprisingly, is comparable to that found from equivalent studies with beta-glucosidases despite the geometric differences at C-2 and the importance of hydrogen bonding at that position. Modes of stabilizing the mannosidase transition state are discussed.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical | |
| pubmed:status |
MEDLINE
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| pubmed:month |
Jun
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| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:day |
17
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| pubmed:volume |
42
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
7195-204
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| pubmed:dateRevised |
2006-11-15
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| pubmed:meshHeading |
pubmed-meshheading:12795616-Amino Acid Sequence,
pubmed-meshheading:12795616-Amino Acid Substitution,
pubmed-meshheading:12795616-Binding Sites,
pubmed-meshheading:12795616-Catalysis,
pubmed-meshheading:12795616-Cellulomonas,
pubmed-meshheading:12795616-Fluorine Radioisotopes,
pubmed-meshheading:12795616-Glycosylation,
pubmed-meshheading:12795616-Hydrogen-Ion Concentration,
pubmed-meshheading:12795616-Kinetics,
pubmed-meshheading:12795616-Mannosidases,
pubmed-meshheading:12795616-Mannosides,
pubmed-meshheading:12795616-Mutagenesis,
pubmed-meshheading:12795616-Nuclear Magnetic Resonance, Biomolecular,
pubmed-meshheading:12795616-Sequence Homology, Amino Acid,
pubmed-meshheading:12795616-Substrate Specificity,
pubmed-meshheading:12795616-Thermodynamics,
pubmed-meshheading:12795616-beta-Mannosidase
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| pubmed:year |
2003
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| pubmed:articleTitle |
Mechanism, mutagenesis, and chemical rescue of a beta-mannosidase from cellulomonas fimi.
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| pubmed:affiliation |
PENCE and Department of Chemistry and Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
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| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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