MacRae I J, Segel I H, Fisher A J
Section of Molecular and Cellular Biology and Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616, USA.
Biochemistry. 2001 Jun 12;40(23):6795-804. doi: 10.1021/bi010367w.
ATP sulfurylase from Penicillium chrysogenum is an allosterically regulated enzyme composed of six identical 63.7 kDa subunits (573 residues). The C-terminal allosteric domain of each subunit is homologous to APS kinase. In the presence of APS, the enzyme crystallized in the orthorhombic space group (I222) with unit cell parameters of a = 135.7 A, b = 162.1 A, and c = 273.0 A. The X-ray structure at 2.8 A resolution established that the hexameric enzyme is a dimer of triads in the shape of an oblate ellipsoid 140 A diameter x 70 A. Each subunit is divided into a discreet N-terminal domain, a central catalytic domain, and a C-terminal allosteric domain. Two molecules of APS bound per subunit clearly identify the catalytic and allosteric domains. The sequence 197QXRN200 is largely responsible for anchoring the phosphosulfate group of APS at the active site of the catalytic domain. The specificity of the catalytic site for adenine nucleotides is established by specific hydrogen bonds to the protein main chain. APS was bound to the allosteric site through sequence-specific interactions with amino acid side chains that are conserved in true APS kinase. Within a given triad, the allosteric domain of one subunit interacts with the catalytic domain of another. There are also allosteric-allosteric, allosteric-N-terminal, and catalytic-catalytic domain interactions across the triad interface. The overall interactions-each subunit with four others-provide stability to the hexamer as well as a way to propagate a concerted allosteric transition. The structure presented here is believed to be the R state. A solvent channel, 15-70 A wide exists along the 3-fold axis, but substrates have access to the catalytic site only from the external medium. On the other hand, a surface "trench" links each catalytic site in one triad with an allosteric site in the other triad. This trench may be a vestigial feature of a bifunctional ("PAPS synthetase") ancestor of fungal ATP sulfurylase.
产黄青霉的ATP硫酸化酶是一种变构调节酶，由六个相同的63.7 kDa亚基（573个残基）组成。每个亚基的C末端变构结构域与APS激酶同源。在APS存在的情况下，该酶在正交空间群（I222）中结晶，晶胞参数为a = 135.7 Å，b = 162.1 Å，c = 273.0 Å。2.8 Å分辨率的X射线结构表明，六聚体酶是一个扁长椭球体形状（直径140 Å×70 Å）的三联体二聚体。每个亚基分为一个离散的N末端结构域、一个中央催化结构域和一个C末端变构结构域。每个亚基结合两个APS分子，清楚地确定了催化结构域和变构结构域。序列197QXRN200在很大程度上负责将APS的磷酸硫酸基团锚定在催化结构域的活性位点。催化位点对腺嘌呤核苷酸的特异性是通过与蛋白质主链的特定氢键建立的。APS通过与真正的APS激酶中保守的氨基酸侧链的序列特异性相互作用与变构位点结合。在给定的三联体中，一个亚基的变构结构域与另一个亚基的催化结构域相互作用。在三联体界面上也存在变构-变构、变构-N末端和催化-催化结构域相互作用。整体相互作用——每个亚基与其他四个亚基相互作用——为六聚体提供了稳定性，也提供了一种传播协同变构转变的方式。这里呈现的结构被认为是R态。沿着三重轴存在一个15 - 70 Å宽的溶剂通道，但底物仅从外部介质进入催化位点。另一方面，一个表面“沟槽”将一个三联体中的每个催化位点与另一个三联体中的变构位点连接起来。这个沟槽可能是真菌ATP硫酸化酶双功能（“PAPS合成酶”）祖先的残留特征。
