Hansen T, Urbanke C, Leppänen V M, Goldman A, Brandenburg K, Schäfer G
Institute of Biochemistry, Medical University of Lübeck, Ratzeburger Allee 160, Lübeck, D-23538, Germany.
Arch Biochem Biophys. 1999 Mar 1;363(1):135-47. doi: 10.1006/abbi.1998.1072.
Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ >> Mn2+ >> Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.
来自嗜热古细菌嗜酸热硫化叶菌（S-PPase）的重组焦磷酸酶已在大肠杆菌中实现异源表达，并能够大量纯化。S-PPase先前被描述为一种四聚体酶，现被证明是一种同六聚体蛋白，其对Mg2+ > Zn2+ > Co2+ >> Mn2+ >> Ni2+、Ca2+具有催化活性。圆二色光谱（CD）和傅里叶变换红外光谱（FTIR）表明，S-PPase与大肠杆菌的PPase（E-PPase）和嗜热栖热菌的PPase（T-PPase）具有相似的整体折叠结构。S-PPase中二级结构元件的相对比例与先前提出的模型相近。S-PPase具有极高的耐热性。即使在95℃时，其催化活性的半衰期仍为2.5小时，在二价阳离子存在的情况下，半衰期会显著延长。催化作用需要每个单体有一个以上的Mg2+，但每个单体有不超过一个Mg2+就足以实现热稳定。S-PPase的解链温度（Tm）值在加入EDTA时为89℃，加入Mg2+时为99℃，加入Mn2+时>100℃；相比之下，E-PPase在加入EDTA时为58℃，加入Mg2+时为84℃，加入Mn2+时为93℃；T-PPase在加入EDTA时为86℃，加入Mg2+时为99℃，加入Mn2+时为96℃。盐酸胍诱导的去折叠遵循一种未知机制，具有双相动力学和一个不稳定的中间体。S-PPase、E-PPase和T-PPase的去折叠曲线与所采用的方法（CD光谱法和荧光法）无关，呈现出S形和单相转变，表明去折叠过程中整体结构发生了变化，这可以用一个双态过程来描述，即折叠的六聚体解离并变性为六个单体。三种PPase各自的ΔGN→D（25℃）值在整个过程中从220到290 kJ/mol不等，两种嗜热PPase的值并没有显著更高。Mg2+的稳定作用ΔΔG（25℃）对于E-PPase为16 kJ/mol，对于S-PPase和T-PPase为5.5 - 8 kJ/mol。
