Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus
Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus
复制标题
DOI：
10.1111/j.1742-4658.2005.05007.x
复制
发表时间：
2005-12-01
期刊：
影响因子：
5.4
通讯作者：
Schönheit, P
中科院分区：
文献类型：
--
作者：
Hansen, T;Schlichting, B;Schönheit, P
最近，cupin型磷酸葡萄糖异构酶被描述为一个新的蛋白质家族，代表了磷酸葡萄糖异构酶进化的一个单独的谱系。以fulgidus Archaeoglobus cPGI （AfcPGI）为模型，通过位点定向诱变评估了cPGI家族中完全保守的8个活性位点残基的重要性。构建并纯化了突变体T63A、G79A、G79L、H80A、H80D、H82A、E93A、E93D、Y95F、Y95K、H136A和Y160F，分析了各突变体对催化和/或金属离子结合以及热稳定性的影响。改型G79A、G79L和Y95F表现出较低的热稳定性。该酶在G79A、G79L、H80A、H80D、E93D、Y95F变异体中的催化效率降低了100倍以上，在T63A、H82A、Y95K、Y160F变异体中的催化效率降低了15倍以上，但在Ni2+饱和条件下对H136A变异体的催化效率保持不变。此外，突变体H80A、H80D、H82A、E93A、E93D的Ni2+含量和Ni2+的表观结合能力降低，几乎完全丧失活性，从而强调了金属离子在催化中的关键作用。结果表明，H80、H82和E93除结合金属离子外，还具有催化作用。E93似乎是AfcPGI的关键催化残基，因为E93A突变体根本没有表现出任何催化活性。
Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by site-directed mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and/or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni2+ saturating conditions. Further, the Ni2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.