Taddei N, Stefani M, Magherini F, Chiti F, Modesti A, Raugei G, Ramponi G
Department of Biochemical Sciences, University of Florence, Italy.
Biochemistry. 1996 Jun 4;35(22):7077-83. doi: 10.1021/bi952900b.
Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.
到目前为止，在分离得到的肌肉和红细胞酰基磷酸酶中，天冬酰胺41（Asn41）、苏氨酸42（Thr42）和苏氨酸46（Thr46）是不变残基。马肌肉酰基磷酸酶的溶液结构表明它们与主要底物结合位点精氨酸23（Arg23）在空间上关系密切。通过寡核苷酸定向诱变制备了几个基因变体（Thr42Ala、Thr46Ala、Asn41Ala、Asn41Ser和Asn41Gln），研究了这些残基的催化和结构作用以及它们对肌肉酰基磷酸酶稳定性的影响。将突变基因克隆并在大肠杆菌中表达，通过亲和层析纯化突变酶，并与野生型酶进行比较研究。Thr42和Thr46突变体的比活性和底物亲和力没有受到显著影响。相反，Asn41突变体的活性残留可忽略不计（与野生型酶相比约为0.05 - 0.15%），尽管其对底物和无机磷酸（一种酶竞争性抑制剂）的结合能力保持不变。根据氢核磁共振光谱和圆二色性结果，所有突变体都诱导出了结构良好的类似天然的二级和三级结构。根据圆二色性数据计算得到的热力学参数表明，在温度和尿素浓度升高时，Thr42突变体的稳定性显著降低。所报道的结果有力地支持了Asn41直接参与酶催化机制，表明Asn41突变体可能是通过负显性方法研究酶生理功能的有用工具。
