Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase.
Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase.
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Asp69 和 Arg310 对 His68（腺苷琥珀酸裂合酶的关键催化残基）pK 的影响。
DOI：
10.1110/ps.072927207
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发表时间：
2007
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：
Sivendran,Sharmila;Segall,MarkL;Rancy,PumtiwittC;Colman,RobertaF
枯草芽孢杆菌的腺苷酸琥珀酸裂解酶 (ASL) 含有三个保守的组氨酸：His68、His89 和 His141，通过亲和标记和定点诱变鉴定为对亚基间催化位点至关重要。野生型 ASL 的 pH-Vmax 曲线呈钟形（pK1= 6.74 和 pK2= 8.28）。只有碱性侧随温度变化，这是组氨酸 pK 的特征。为了确定酶-底物复合物中 pK2 的决定因素，我们替换了结构中靠近 His68（但不是 His89 或 His141）的两个位置的残基。与pH 7.0下1.75μmol腺苷酸琥珀酸反应/min/mg野生型酶的比活性相比，突变酶D69E、D69N、R310Q和R310K的比活性分别为0.40、0.04、0.00083和0.10。虽然 D69E 的腺苷酸琥珀酸 Km 与野生型 ASL 相似，但 D69N 和 R310K 的 Km 略有增加，而 R310Q 的 Km 增加了 11 倍。突变酶的分子量或二级结构没有显着变化。主要变化在于 pH-Vmax 曲线：D69E 突变体的 pK2 为 8.48，D69N 中的 pK2 降至 7.83，这表明需要近端负电荷来维持野生型酶观察到的高 pKof 8.28（归因于 His68）。类似地，R310Q 的 pK2 (7.33) 有所降低，而 R310K 的 pK2 (8.24) 几乎没有变化。这些结果表明 Asp69 与 His68 相互作用，Arg310 与 Asp69 的 β-羧酸盐相互作用并定向，并且 His68 必须被质子化才能使 ASL 具有活性。
Adenylosuccinate lyase (ASL) ofBacillus subtiliscontains three conserved histidines, His68, His89, and His141, identified by affinity labeling and site‐directed mutagenesis as critical to the intersubunit catalytic site. The pH‐Vmaxprofile for wild‐type ASL is bell‐shaped (pK1= 6.74 and pK2= 8.28). Only the alkaline side changes with temperature, characteristic of histidine pKs. To identify determinants of pK2in the enzyme‐substrate complex, we replaced residues at two positions close to His68(but not to His89or His141) in the structure. Compared with the specific activity of 1.75μmol adenylosuccinate reacting/min/mg of wild‐type enzyme at pH 7.0, mutant enzymes D69E, D69N, R310Q, and R310K exhibit specific activities of 0.40, 0.04, 0.00083, and 0.10, respectively. While D69E has aKmfor adenylosuccinate similar to that of wild‐type ASL, D69N and R310K exhibit modest increases inKm, and R310Q has an 11‐fold increase inKm. The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH‐Vmaxprofile: pK2is 8.48 for the D69E mutant and is decreased to 7.83 in D69N, suggesting a proximal negative charge is needed to maintain the high pKof 8.28 observed for wild‐type enzyme and attributed to His68. Similarly, R310Q exhibits a decrease in its pK2(7.33), whereas R310K shows little change in pK2(8.24). These results suggest that Asp69interacts with His68, that Arg310interacts with and orients theβ‐carboxylate of Asp69, and that His68must be protonated for ASL to be active.