Palenchar Jennifer Brosius, Crocco Jennifer M, Colman Roberta F
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Protein Sci. 2003 Aug;12(8):1694-705. doi: 10.1110/ps.0303903.
Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR). Several point mutations in the gene encoding the enzyme have been implicated in human disease. Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency. Site-directed mutagenesis was utilized to construct amino acid substitutions in B. subtilis adenylosuccinate lyase; Met(10), Ile(123), and Thr(367) were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli. These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters. The major deviations from the wild-type-like behavior were observed upon biophysical characterization. All of these enzymes with amino acid replacements are associated with marked thermal instability. I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation. T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis. Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired. The enzyme instability was found to be lessened by subunit complementation with the wild-type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients. Residues Met(10), Ile(123), and Thr(367) appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme.
腺苷酸琥珀酸裂解酶是一种同四聚体，在嘌呤的从头合成中催化两个不同的反应：腺苷酸琥珀酸和琥珀酰氨基咪唑甲酰胺核苷酸（SAICAR）的裂解。编码该酶的基因中的几个点突变与人类疾病有关。枯草芽孢杆菌腺苷酸琥珀酸裂解酶被用作模型系统，构建与严重人类腺苷酸琥珀酸裂解酶缺乏相关的突变。利用定点诱变在枯草芽孢杆菌腺苷酸琥珀酸裂解酶中构建氨基酸替换；分别将Met(10)、Ile(123)和Thr(367)替换为Leu、Trp和Arg，并在大肠杆菌中表达改变后的酶。发现这些含有氨基酸替换的纯化酶具有相当大的催化活性，并且其动力学参数变化相对较小。在生物物理表征时观察到与野生型样行为的主要偏差。所有这些具有氨基酸替换的酶都与明显的热不稳定性有关。I123W腺苷酸琥珀酸裂解酶在圆二色光谱中表现出显著变化，并且在天然凝胶电泳图谱中显示出一些蛋白质聚集的迹象。T367R在四级结构水平上也表现出改变，这在天然凝胶电泳中有所反映。实验结果与同源建模相结合表明，改变后的酶主要在结构上受损。在温和条件下，通过与野生型酶的亚基互补发现酶的不稳定性降低；这些研究可能对杂合患者体内腺苷酸琥珀酸裂解酶的行为有影响。残基Met(10)、Ile(123)和Thr(367)似乎位于酶的对维持稳定、功能性酶所需的结构完整性很重要的区域。
