Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis:: structural, biochemical, enzymatic and stability studies
Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis:: structural, biochemical, enzymatic and stability studies
复制标题
DOI：
10.1007/s00775-006-0102-0
复制
发表时间：
2006-06-01
影响因子：
3
通讯作者：
Martins, LO
中科院分区：
文献类型：
--
作者：
Durao, P;Bento, I;Martins, LO
定点诱变已被用于用亮氨酸和苯丙氨酸残基取代科塔漆酶中的Met 502。M502 L和M502 F突变体与野生型科塔的X射线结构比较显示，T1铜位点的几何形状以及蛋白质的整体折叠得以保持。T1位点的弱的所谓轴向配体的替换导致氧化还原电位相对于野生型酶（E-0=455 mV）增加约100 mV。然而，M502 L突变体对于所有测试的底物表现出k（cat）值的2倍至4倍降低，并且M502 F中的催化活性甚至更严重地受损;与野生型酶相比，10%的活性和0.15-0.05%的非酚底物和酚底物的活性。T1铜耗尽是失活的关键事件，因此它是野生型和突变蛋白质的热力学稳定性的决定因素。虽然在野生型酶的三级结构的展开是一个两态的过程，显示在盐酸胍浓度为4.6 M和自由能交换在水中的10千卡/摩尔的中点，这两个突变体酶的展开显然不是一个两态的过程。在1.9 M盐酸胍时，一半的分子处于中间构象，仅略低于天然状态（约1.4 kcal/mol）。从结构、催化和稳定性的角度来看，T1铜中心在科塔漆酶活性的调节中显然起着关键作用。
Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E-0=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k (cat) values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15-0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.