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dc.contributor.authorLiu, Sheng-Wenen_US
dc.contributor.authorChen, Chao-Shengen_US
dc.contributor.authorChang, Shih-Shenen_US
dc.contributor.authorMong, Kwok-Kong Tonyen_US
dc.contributor.authorLin, Chun-Hungen_US
dc.contributor.authorChang, Cheng-Wenen_US
dc.contributor.authorTang, Chuan Yien_US
dc.contributor.authorLi, Yaw-Kuenen_US
dc.date.accessioned2014-12-08T15:10:08Z-
dc.date.available2014-12-08T15:10:08Z-
dc.date.issued2009-01-13en_US
dc.identifier.issn0006-2960en_US
dc.identifier.urihttp://dx.doi.org/10.1021/bi801529ten_US
dc.identifier.urihttp://hdl.handle.net/11536/7749-
dc.description.abstractFucosylated glycoconjugates have critical roles in biological processes, but a limited availability Of alpha-L-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Bronsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(1g) = -0.93, but that for k(cat) is biphasic, with beta(1g) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of Pig for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter Pig value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that ASp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.en_US
dc.language.isoen_USen_US
dc.titleIdentification of Essential Residues of Human alpha-L-Fucosidase and Tests of Its Mechanismen_US
dc.typeArticleen_US
dc.identifier.doi10.1021/bi801529ten_US
dc.identifier.journalBIOCHEMISTRYen_US
dc.citation.volume48en_US
dc.citation.issue1en_US
dc.citation.spage110en_US
dc.citation.epage120en_US
dc.contributor.department應用化學系zh_TW
dc.contributor.departmentDepartment of Applied Chemistryen_US
dc.identifier.wosnumberWOS:000262265900012-
dc.citation.woscount12-
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