標題: Identification of Essential Residues of Human alpha-L-Fucosidase and Tests of Its Mechanism
作者: Liu, Sheng-Wen
Chen, Chao-Sheng
Chang, Shih-Shen
Mong, Kwok-Kong Tony
Lin, Chun-Hung
Chang, Cheng-Wen
Tang, Chuan Yi
Li, Yaw-Kuen
應用化學系
Department of Applied Chemistry
公開日期: 13-一月-2009
摘要: Fucosylated 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.
URI: http://dx.doi.org/10.1021/bi801529t
http://hdl.handle.net/11536/7749
ISSN: 0006-2960
DOI: 10.1021/bi801529t
期刊: BIOCHEMISTRY
Volume: 48
Issue: 1
起始頁: 110
結束頁: 120
顯示於類別:期刊論文


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  1. 000262265900012.pdf