1EGN

CELLOBIOHYDROLASE CEL7A (E223S, A224H, L225V, T226A, D262G) MUTANT

1EGN の概要

• エントリーDOI: 10.2210/pdb1egn/pdb
• 関連するPDBエントリー: 1DY4 1EK7 1I5M 4CEL 7CEL
• 分子名称: 1,4-BETA-D-GLUCAN CELLOBIOHYDROLASE CEL7A, 2-acetamido-2-deoxy-beta-D-glucopyranose, COBALT (II) ION, ... (4 entities in total)
• 機能のキーワード: hydrolase, glycosidase, cellulase, cellulose degradation, glycoprotein, glycosylated protein, ph-mutant
• 由来する生物種: Hypocrea jecorina
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 46330.75

構造登録者

Stahlberg, J.,Harris, M.,Jones, T.A. (登録日: 2000-02-16, 公開日: 2001-05-16, 最終更新日: 2024-10-09)

主引用文献

Becker, D.,Braet, C.,Brumer III, H.,Claeyssens, M.,Divne, C.,Fagerstrom, B.R.,Harris, M.,Jones, T.A.,Kleywegt, G.J.,Koivula, A.,Mahdi, S.,Piens, K.,Sinnott, M.L.,Stahlberg, J.,Teeri, T.T.,Underwood, M.,Wohlfahrt, G.
Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant.
Biochem.J., 356:19-30, 2001

PubMed Abstract: The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

PubMed: 11336632
DOI: 10.1042/0264-6021:3560019

実験手法

X-RAY DIFFRACTION (1.6 Å)