Identification of essential residues of human alpha-L-fucosidase and tests of its mechanism.
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.
Liu
Sheng-Wen
SW
Department of Applied Chemistry, National Chiao Tung University, Institute of Biological Chemistry, Academia Sinica, Hsinchu, Taiwan.
Chen
Chao-Sheng
CS
Chang
Shih-Shen
SS
Mong
Kwok-Kong Tony
KK
Lin
Chun-Hung
CH
Chang
Cheng-Wen
CW
Tang
Chuan Yi
CY
Li
Yaw-Kuen
YK
D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D001386:Azides; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005644:alpha-L-Fucosidase