Chiti F, Magherini F, Taddei N, Ilardi C, Stefani M, Bucciantini M, Dobson C M, Ramponi G
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, UK.
Protein Eng. 1998 Jul;11(7):557-61. doi: 10.1093/protein/11.7.557.
An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.
对五个酰基磷酸酶保守赖氨酸残基进行了寡核苷酸定向诱变研究，以评估它们在酶活性位点形成中的可能参与情况及其对酶构象稳定性的贡献。该研究旨在消除红细胞同工酶晶体结构中与赖氨酸32和68结合的硫酸根离子（一种酶竞争性抑制剂）的存在所产生的歧义。此外，先前的动力学研究表明存在pKa = 7.9和11的残基，初步鉴定为两个赖氨酸。所研究突变体的动力学参数与野生型酶的动力学参数无显著差异，表明没有赖氨酸残基参与催化或底物结合。此外，通过圆二色性进行的热变性和尿素变性实验表明，突变的赖氨酸残基在酶结构稳定中不发挥重要作用，因为去稳定化能量平均为1.40 kJ/mol。这些结果与其他蛋白质的结果一致，表明赖氨酸残基对天然结构的稳定性贡献很小。
