Identification of Essential Residues of Human α-l-Fucosidase and Tests of Its Mechanism

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• 作者：Sheng-Wen Liu ; Chao-Sheng Chen ; Shih-Shen Chang ; Kwok-Kong Tony Mong ; Chun-Hung Lin ; Cheng-Wen Chang ; Chuan Yi Tang ; Yaw-Kuen Li
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：January 13, 2009
• 年：2009
• 卷：48
• 期：1
• 页码：110-120
• 全文大小：538K
• 年卷期：v.48,no.1(January 13, 2009)
• ISSN：1520-4995

文摘

Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of α-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 Brønsted plot for k_cat/K_m for the E289G mutant is linear with β_lg = −0.93, but that for k_cat is biphasic, with β_lg for poor substrates being −0.88 and for activated substrates being −0.11. The small magnitude of β_lg for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter β_lg value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp²²⁵ functions as a nucleophile and Glu²⁸⁹ as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and ¹H NMR spectral analysis confirmed the formation of β-fucosyl azide and α-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu²⁸⁹, an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.