1ZVK

Structure of Double mutant, D164N, E78H of Kumamolisin-As

1ZVK の概要

• エントリーDOI: 10.2210/pdb1zvk/pdb
• 関連するPDBエントリー: 1S1O 1S1U 1SN7 1ZVK
• 分子名称: kumamolisin-As, CALCIUM ION (3 entities in total)
• 機能のキーワード: d164n, e78h, hydrolase
• 由来する生物種: Alicyclobacillus sendaiensis
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 72315.68

構造登録者

Li, M.,Wlodawer, A.,Gustchina, A.,Nakayama, T. (登録日: 2005-06-02, 公開日: 2006-05-23, 最終更新日: 2024-02-14)

主引用文献

Okubo, A.,Li, M.,Ashida, M.,Oyama, H.,Gustchina, A.,Oda, K.,Dunn, B.M.,Wlodawer, A.,Nakayama, T.
Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site.
Febs J., 273:2563-2576, 2006

PubMed Abstract: Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.

PubMed: 16704427
DOI: 10.1111/j.1742-4658.2006.05266.x

実験手法

X-RAY DIFFRACTION (2.04 Å)