Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis.
Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis.
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DOI：
10.2210/pdb1hmu/pdb
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发表时间：
2001-01
期刊：
影响因子：
2.9
通讯作者：
W. Huang;L. Boju;L. Tkalec;H. Su;H. Yang;N. Gunay;R. Linhardt;Y. S. Kim;A. Matte;M. Cygler-M.
中科院分区：
文献类型：
--
作者：
W. Huang;L. Boju;L. Tkalec;H. Su;H. Yang;N. Gunay;R. Linhardt;Y. S. Kim;A. Matte;M. Cygler-M.
结合至硫酸皮肤素六糖（DS（hexa））、四糖（DS（tetra））和透明质酸四糖（HA（tetra））的肝素黄杆菌软骨素AC裂解酶（chondroitinase AC; EC 4.2.2.5）的晶体结构已经分别在2.0、2.0和2.1 A分辨率下被精制。与硫酸软骨素四糖（CS（tetra））结合的AC裂解酶的Tyr 234 Phe突变体的结构也被确定为2.3 A分辨率。对于这些复合物中的每一个，在电子密度图中可以看到四个（DS（六）和CS（四））或两个（DS（四）和HA（四））有序糖。裂解酶AC DS（hexa）和CS（tetra）复合物显示在一个狭窄而浅的蛋白质通道内的四个亚位点（-2，-1，+1和+2）结合。我们认为，亚位点-2和-1一起代表底物识别区域，+1是催化亚位点，+1和+2一起代表产物释放区域。推定的催化位点位于底物识别区和产物释放区之间，在+1亚位点进行催化。催化位点附近的四个残基His 225、Tyr 234、Arg 288和Glu 371一起形成催化四联体。突变His 225 Ala、Tyr 234 Phe、Arg 288 Ala和Arg 292 Ala仅显示Arg 292 Ala突变体的残余活性。结构数据表明，Arg 292主要参与半乳糖胺的N-乙酰基和硫酸酯部分的识别，但不直接参与催化。去除在+1亚位点处连接到葡萄糖醛酸的C-5的质子的一般碱的候选物是Tyr 234，其可以在催化过程中瞬时去质子化，或His 225。酪氨酸234是使离去基团质子化的候选者。精氨酸288可能有助于催化过程中烯醇化物阴离子中间体的电荷中和和稳定。
The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area. The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.