Identification of Essential Residues of Human α-<scp>l</scp>-Fucosidase and Tests of Its Mechanism
In: Biochemistry, Jg. 48 (2008-12-12), S. 110-120
Online
unknown
Zugriff:
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 Brønsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(lg) = -0.93, but that for k(cat) is biphasic, with beta(lg) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of beta(lg) for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter beta(lg) 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.
Titel: |
Identification of Essential Residues of Human α-<scp>l</scp>-Fucosidase and Tests of Its Mechanism
|
---|---|
Autor/in / Beteiligte Person: | Chen, Chao-Sheng ; Chang, Shih-Shen ; Kwok-Kong Tony Mong ; Lin, Chun-Hung ; Chang, Cheng-Wen ; Liu, Sheng-Wen ; Chuan Yi Tang ; Li, Yaw-Kuen |
Link: | |
Zeitschrift: | Biochemistry, Jg. 48 (2008-12-12), S. 110-120 |
Veröffentlichung: | American Chemical Society (ACS), 2008 |
Medientyp: | unknown |
ISSN: | 1520-4995 (print) ; 0006-2960 (print) |
DOI: | 10.1021/bi801529t |
Schlagwort: |
|
Sonstiges: |
|