Structure of the two-subsite β-D-xyloidase from Selenomonas ruminantium in complex with 1,3-bis [tris(hydroxymethyl)methylamino] propane
Structure of the two-subsite β-D-xyloidase from Selenomonas ruminantium in complex with 1,3-bis [tris(hydroxymethyl)methylamino] propane
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DOI：
10.1016/j.abb.2008.03.007
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发表时间：
2008-06-01
影响因子：
3.9
通讯作者：
Wawrzak, Zdzislaw
中科院分区：
文献类型：
--
作者：
Brunzelle, Joseph S.;Jordan, Douglas B.;Wawrzak, Zdzislaw
采用x射线晶体学（1.3埃分辨率）测定了反刍硒单胞菌与竞争抑制剂1,3-二[三（羟甲基）甲胺]丙烷（BTP）配合物中高效催化β -木糖苷酶的三维结构。抑制剂与蛋白之间的大多数H键发生在亚位-1内，包括E186的羧基与BTP的N基之间的H键。BTP的另一个N位于K99附近的子站点+1。E186 （pK(a) 7.2）作为催化酸。1/K-i（(BTP)）的pH值（6-10）呈钟形，E186和抑制剂组的酸性端pKa值为6.8和7.8，抑制剂组的碱性端pKa值为9.6。突变K99A消除了pK(a) 7.8，强烈表明BTP单位结合了重阴离子酶D14(-)E186（-）。沉积平衡实验估计k -d（[二聚体](2)/[四聚体]）为7 × 10(-9) m，当四聚体/二聚体的比例从0.0028变化到26时，k（cat）和k(cat)/ k -m值也相近，表明二聚体和四聚体是同样活跃的形式。(C) 2008爱思唯尔公司版权所有。
The three-dimensional structure of the catalytically efficient beta-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis [tris(hydroxymethyl)methylamino] propane (BTP) was determined by using X-ray crystallography (1.3 angstrom resolution). Most H bonds between inhibitor and protein occur within subsite-1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies SUbsite +1 near K99. E186 (pK(a) 7.2) serves as catalytic acid. The pH (6-10) profile for 1/K-i((BTP)) is bell-shaped with pKa's 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.6 on the basic limb assigned to inhibitor. Mutation K99A eliminates pK(a) 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14(-)E186(-). A sedimentation equilibrium experiment estimates a K-d ([dimer](2)/[tetramer]) of 7 x 10(-9) M. Similar k(cat) and k(cat)/K-m values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms. (C) 2008 Elsevier Inc. All rights reserved.