Kinetic study on the active site topology of a Family 3 β-glucosidase
Dr. Yaw-Kuen Li
根據燕麥β-葡萄糖甘酵素分子模擬及其他物種之葡萄糖甘酵素胺基酸序列比對的結果，我們以定點突變技術建構並純化一系列突變酵素E132G、D275N、 D458N、E469Q、E473Q、E473G、E473D、E473C、D537N以進行研究。由以下的結果推論E473為general acid/base.
(1) 由動力學數據發現E473G和wild-type比較，其對2,4-DNPG 之kcat 減少約3000倍。(2)而且在pH 5.0~9.0之間kcat並沒有很大的變化，和wild-type不一樣 ，表示E473突變後acid/base catalyst 已經不存在。(3)E473G和2,4-DNPGlu反應之活性因添加azide而大幅增加，且生成β-glucosyl azide.(4)E473G 和2- carboxyphenyl ß-glucoside反應，其反應速率和4-carboxyphenyl ß-glucoside1比較明顯增加，顯示E473為general acid/base.
A β-glucosidase gene from Flavobacterium meningosptium has been cloned and expressed in E.coli. In this study, we investigated the catalytic role of the conserved sequences by site-directed mutagenesis. Several point of mutants, including E132, D275, D458, E469, and E473 have been constructed . kinetic studies on these mutants were compared with that of wild type. Among the mutants, E473 was identified with general acid/base catalyst. Based on the following results： (1) The catalytic activity of E473 toward 2,4-DNPG was reduced 3000-fold. (2) Unlike the the bell-shaped pH profile of wild type, the kcat values were virtually invariant with pH over the range of 5.0-9.0, indicating the general acid/base catalyst was absent on E473G mutant . (3) The activity of E473G towards 2,4-DNPG was largely enhanced by the addition of anion such as azide. Glucosyl azide was found to be the product. (4) A large rate activation on the case of E473G hydrolyzed 2-carboxyphenyl glucoside strongly suggested that E473 is the acid/base catalyst. When the amino acid residue responsible for substrate binding were mutated , the Km values were mostly unchanged, whereas the kcat values were reduced by 10--2400-fold. For these mutants with large Km perturbation , Circular Dichroism spectroscopy study revealed that no significant (secondary) structure change was observed indicating the large Km values was likely during the purification resulted from the loss of hydrogen bond between enzyme and substrate.
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