Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate
Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate
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DOI：
10.1074/jbc.m110.101071
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发表时间：
2010-06-25
影响因子：
4.8
通讯作者：
Cygler, Miroslaw
中科院分区：
文献类型：
--
作者：
Shaya, David;Zhao, Wenjing;Cygler, Miroslaw
肝素酶II（HepII）是一种85 kDa的二聚酶，通过β-消除机制解聚肝素和硫酸乙酰肝素糖胺聚糖。最近，我们确定了肝素足杆菌（以前称为肝素黄杆菌）与肝素二糖产物复合的HepII的晶体结构，并确定了其活性位点的位置。在这里，我们提出了HepII与硫酸乙酰肝素二糖产物复合的结构，证明了相同的结合/活性位点是负责两个糖醛酸差向异构体含有底物的降解。关键的酶促步骤涉及从糖醛酸的C5碳（手性中心）去除质子，这对从单个活性位点中的环的任一侧提取质子提出了拓扑挑战。我们已经确定了三个潜在的活性位点残基等距C5和位于两侧的糖醛酸产物，并确定其在催化中的作用，使用一组定义的四糖底物。HepII H202 A/Y257 A突变体失去了对两种底物的活性，我们确定了其与硫酸乙酰肝素衍生的四糖复合的晶体结构。基于各种突变体的动力学特性和酶-底物复合物的结构，我们提出了参与催化的残基及其具体作用。
Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a beta-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.