Waratrujiwong Teerawit, Krebs Bernt, Spener Friedrich, Visoottiviseth Pornsawan
Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand.
FEBS J. 2006 Apr;273(8):1649-59. doi: 10.1111/j.1742-4658.2006.05179.x.
Purple acid phosphatases (PAPs) from sweet potato (sp) have been classified on the basis of their primary structure and the dinuclear metal center into isoforms spPAP1 [Fe(III)-Zn(II)] and spPAP2 [Fe(III)-Mn(II)]; for spPAP3 only the cDNA is known. With the aim of unraveling the character of the dinuclear metal center we report here the characterization of this isoform at the protein level. We cloned spPAP3 cDNA in a baculovirus and overexpressed this enzyme in Sf9 insect cells. Preparation of recombinant spPAP3 in two steps afforded pure enzyme with yields of 4.5 mg.L(-1) culture medium. This enzyme is a dimeric, disulfide-linked PAP of 110 kDa, similar to known PAP isoforms from higher plants. Enzymatic studies and spectroscopic properties (max. absorption at 550-565 nm) indicated a diiron enzyme; quantitative and semiquantitative metal analysis using ICP-OES and TOF-SIMS, respectively, revealed the presence of only iron in purified spPAP3. Metal replacement in the second metal-binding site upon preparation of the semiapo-enzyme with Fe(II), Zn(II), or Mn(II) showed highest activities with Fe(II). The data show that recombinant spPAP3 has a diiron metal center. Site-directed mutagenesis was conducted to check catalytic efficiency at the atomic level. Tyr291 at the substrate-binding site in spPAP3 was mutated to His and Ala, the respective residues found in spPAP1 and spPAP2. Kinetic analysis showed that conversion of Tyr291 to His further optimized the performance of this protein as a diiron enzyme, whereas the Ala mutation weakened the catalytic efficiency regardless of the metal present in the second binding site.
甘薯（sp）中的紫色酸性磷酸酶（PAPs）已根据其一级结构和双核金属中心分为同工型spPAP1 [Fe(III)-Zn(II)] 和spPAP2 [Fe(III)-Mn(II)]；对于spPAP3，仅知道其cDNA。为了阐明双核金属中心的特性，我们在此报告该同工型在蛋白质水平上的表征。我们将spPAP3 cDNA克隆到杆状病毒中，并在Sf9昆虫细胞中过表达该酶。通过两步制备重组spPAP3，得到了纯酶，产量为4.5 mg·L⁻¹ 培养基。该酶是一种110 kDa的二聚体、二硫键连接的PAP，与高等植物中已知的PAP同工型相似。酶学研究和光谱性质（最大吸收波长在550 - 565 nm）表明这是一种双铁酶；分别使用电感耦合等离子体发射光谱法（ICP - OES）和飞行时间二次离子质谱法（TOF - SIMS）进行的定量和半定量金属分析显示，纯化的spPAP3中仅存在铁。在用Fe(II)、Zn(II)或Mn(II)制备半脱辅基酶时，第二金属结合位点的金属置换显示，Fe(II)具有最高活性。数据表明重组spPAP3具有双铁金属中心。进行了定点诱变以在原子水平上检查催化效率。spPAP3底物结合位点的Tyr291被突变为His和Ala，这是在spPAP1和spPAP2中发现的相应残基。动力学分析表明，将Tyr291转换为His进一步优化了该蛋白作为双铁酶的性能，而Ala突变则削弱了催化效率，无论第二结合位点中存在何种金属。
