1KR1

Hevamine Mutant D125A/E127A in Complex with Tetra-NAG

1KR1 の概要

• エントリーDOI: 10.2210/pdb1kr1/pdb
• 関連するPDBエントリー: 1hvq 1kqy 1kqz 1kr0 1llO 2hvm
• 分子名称: Hevamine A, 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, SULFATE ION, ... (4 entities in total)
• 機能のキーワード: hydrolase, chitinase/lysozyme
• 由来する生物種: Hevea brasiliensis
• 細胞内の位置: Vacuole: p23472
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 30397.99

構造登録者

Rozeboom, H.J.,Dijkstra, B.W. (登録日: 2002-01-08, 公開日: 2002-01-23, 最終更新日: 2024-11-20)

主引用文献

Bokma, E.,Rozeboom, H.J.,Sibbald, M.,Dijkstra, B.W.,Beintema, J.J.
Expression and Characterization of Active Site Mutants of Hevamine, a Chitinase from the Rubber Tree Hevea brasiliensis.
Eur.J.Biochem., 269:893-901, 2002

PubMed Abstract: Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.

PubMed: 11846790
DOI: 10.1046/j.0014-2956.2001.02721.x

実験手法

X-RAY DIFFRACTION (2 Å)