Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis.
Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis.
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Gln212、Asn270 和 Arg301 对于枯草芽孢杆菌腺苷琥珀酸裂解酶的催化作用至关重要。
DOI：
10.1021/bi0494774
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Colman,RobertaF
中科院分区：
文献类型：
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作者：
Segall,MarkL;Colman,RobertaF
在来自枯草芽孢杆菌的腺苷酸琥珀酸裂合酶中，Gln212、Asn270 和 Arg301 是保守的，并且位于靠近对接的腺苷酸琥珀酸的琥珀酰基部分。我们构建了突变酶，其中Gln212被Glu和Met替换，Asn270被Asp和Leu替换，Arg301被Gln或Lys替换。野生型和突变型酶在大肠杆菌中表达并纯化至均质。与野生型相比，Q212M以及270和301突变酶的比活性降低了3000倍以上。只有 Q212E 保留了足够的活性来测定其动力学参数：与野生型酶相比，Vmax 降低约 1000 倍，Km 增加 6 倍。其他突变体的腺苷酸琥珀酸结合研究揭示了亲和力大大减弱，这导致但并不能完全解释活性丧失。圆二色光谱和光散射光度测定表明，这些突变酶在二级结构或亚基缔合状态方面与野生型酶没有太大差异。成对的失活突变酶的孵育导致通过亚基互补重建一些功能位点，恢复野生型酶高达 25% 的比活性。仅当两个突变由不同的亚基贡献给活性位点时，才会发生亚基互补。因此，将 Q212E 与 N270L 酶混合产生的比活性约为野生型酶的 20%，而将 Q212M 与 R301K 酶混合则没有恢复活性。在计算机模型的支持下，本文提出的研究表明，Gln212、Asn270 和 Arg301 对于腺苷酸琥珀酸裂合酶的催化作用是不可或缺的，并且可能与底物 5-氨基咪唑-4(N-琥珀酰酰胺)核糖核苷酸和腺苷酸琥珀酸的羧酸根阴离子发生非共价相互作用，从而优化它们的结合方向。
In adenylosuccinate lyase fromBacillus subtilis, Gln212, Asn270, and Arg301are conserved and located close to the succinyl moiety of docked adenylosuccinate. We constructed mutant enzymes with Gln212replaced by Glu and Met, Asn270by Asp and Leu, and Arg301by Gln or Lys. The wild-type and mutant enzymes were expressed inEscherichia coliand purified to homogeneity. The specific activities of the Q212M and the 270 and 301 mutant enzymes were decreased more than 3000-fold as compared to the wild type. Only Q212E retained sufficient activity for determination of its kinetic parameters:Vmaxwas decreased ∼1000-fold, andKmwas increased 6-fold, as compared to the wild-type enzyme. Adenylosuccinate binding studies of the other mutants revealed greatly weakened affinities that contributed to, but did not account entirely for, the loss of activity. These mutant enzymes did not differ greatly from the wild-type enzyme in secondary structure or subunit association state, as shown by circular dichroism spectroscopy and light-scattering photometry. Incubation of pairs of inactive mutant enzymes led to reconstitution of some functional sites by subunit complementation, with recovery of up to 25% of the specific activity of the wild-type enzyme. Subunit complementation occurs only if the two mutations are contributed to the active site by different subunits. Thus, mixing Q212E with N270L enzyme yielded a specific activity of ∼20% of the wild-type enzyme, while mixing Q212M with R301K enzyme did not restore activity. As supported by computer modeling, the studies presented here indicate that Gln212, Asn270, and Arg301are indispensable to catalysis by adenylosuccinate lyase and probably interact noncovalently with the carboxylate anions of the substrates 5-aminoimidazole-4(N-succinylocarboxamide)ribonucleotide and adenylosuccinate, optimizing their bound orientations.