Banci L, Benedetto M, Bertini I, Del Conte R, Piccioli M, Viezzoli M S
Department of Chemistry, University of Florence, Italy.
Biochemistry. 1998 Aug 25;37(34):11780-91. doi: 10.1021/bi9803473.
Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that no cooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobic residues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structure of the protein, thus producing a soluble monomeric form. However, this monomeric form was found to have an activity lower than that of the native dimeric species (10%). To answer the fundamental question of the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determined the solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamental issue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the active center. The three-dimensional solution structure of this 153-residue monomeric form of SOD (16 kDa) has been determined using distance and dihedral angle constraints obtained from 13C, 15N triple-resonance NMR experiments. The solution structure is represented by a family of 36 structures, with a backbone rmsd of 0.81 +/- 0.13 A over residues 3-150 and of 0.56 +/- 0.08 A over residues 3-49 and 70-150. This structure has been compared with the available X-ray structures of reduced SODs as well as with the oxidized form of human and bovine isoenzymes. The structure contains the classical eight-stranded Greek key beta-barrel. In general, the backbone and the metal sites are not affected much by the monomerization, except in the region involved in the subunit-subunit interface in the dimeric protein, where a large disorder is present. Significative changes are observed in the conformation of the electrostatic loop, which forms one side of the active site channel and which is fundamental in determining the optimal electrostatic potential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymatic reaction under nonsaturating conditions. In the present monomer, its conformation is less favorable for the diffusion of the substrate to the reaction site. The structure of the copper center is well-defined; copper(I) is coordinated to three histidines, at variance with copper(II) which is bound to four histidines. The hydrogen atom which binds the histidine nitrogen detached from copper(I) is structurally identified.
铜锌超氧化物歧化酶是一种二聚体酶，研究表明其二聚体的两个亚基之间不存在协同作用。在界面区域用两个谷氨酸取代两个疏水残基苯丙氨酸50和甘氨酸51，破坏了蛋白质的四级结构，从而产生了一种可溶的单体形式。然而，发现这种单体形式的活性低于天然二聚体（10%）。为了回答四级结构在超氧化物歧化酶催化过程中的作用这一基本问题，我们通过核磁共振光谱法确定了还原单体突变体的溶液结构。关于酶促机制的另一个基本问题是还原态铜（活性中心）的配位。利用从13C、15N三共振核磁共振实验获得的距离和二面角约束，确定了这种153个残基单体形式的超氧化物歧化酶（16 kDa）的三维溶液结构。溶液结构由36个结构组成的家族表示，在3 - 150位残基上主链均方根偏差为0.81±0.13 Å，在3 - 49位和70 - 150位残基上为0.56±0.08 Å。已将该结构与还原态超氧化物歧化酶的现有X射线结构以及人和牛同工酶的氧化形式进行了比较。该结构包含经典的八链希腊钥匙β桶。一般来说，主链和金属位点受单体化影响不大，除了在二聚体蛋白质中涉及亚基 - 亚基界面的区域存在较大无序。在静电环的构象中观察到显著变化，静电环形成活性位点通道的一侧，对于在非饱和条件下驱动超氧阴离子到达铜位点（酶促反应的限速步骤）确定最佳静电势至关重要。在当前单体中，其构象不利于底物扩散到反应位点。铜中心的结构明确；铜（I）与三个组氨酸配位，这与铜（II）与四个组氨酸结合不同。结合从铜（I）脱离的组氨酸氮的氢原子在结构上已被确定。
