| Summary: | 博士 === 國立交通大學 === 應用化學系所 === 94 === A gene from Trichoderma koningii G-39 encoding an enzyme with α-L-arabinofuranosidase/β-xylosidase (Abf; member of the GH54 family) activity was expressed in Pichia pastoris. The recombinant wild-type enzyme and mutants were purified to > 90% homogeneity by cation-exchange chromatography. Extensive mutagenesis of 24 conserved Glu and Asp residues of family 54 was performed. The kcat values of the D221N and D299N were 7000- and 1300-fold lower than that of the wild-type Abf, respectively, while E223Q was completely inactive. These results are consistent with implications from the Aspergillus kawachii α-L-arabinofuranosidase three-dimensional structure. This structure indicates that E223 of T. koningii Abf function as a nucleophile and D299 as a general acid/base catalyst for the enzymatic reaction. D221 in Abf is significant for substrate binding. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyzes various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-α-L-arabinofuranosides catalyzed by wild-type Abf, a relatively small Brønsted constant, βlg = – 0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reactions for substrates (where the leaving phenols have pKa values >= 5.15) is the dearabinosylation step. Further kinetic studies with D299G mutant revealed that the catalytic activity of this mutant depended largely on the pKa values (> 6) of leaving phenols, with βlg = –1.3. This indicated that the rate-limiting step became arabinosylation step when D299G was employed. This kinetic outcome supports the idea that D299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.
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