Characterization of the putative GTP-binding site residues of Escherichia coli adenylosuccinate synthetase by site-directed mutagenesis.
Characterization of the putative GTP-binding site residues of Escherichia coli adenylosuccinate synthetase by site-directed mutagenesis.
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通过定点诱变表征大肠杆菌腺苷酸琥珀酸合成酶的假定 GTP 结合位点残基。
DOI：
10.1006/abbi.1994.1195
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发表时间：
1994
影响因子：
3.9
通讯作者：
Fromm,HJ
中科院分区：
文献类型：
--
作者：
Kang,C;Fromm,HJ
腺苷酸琥珀酸合成酶在其GTP结合结构域中包含与其他G蛋白同源的氨基酸序列。这种同源性包括富含甘氨酸的磷酸结合环GXXXXGK和鸟嘌呤特异性结合区（N/T/Q）KXD;然而，其他G蛋白和腺苷酸琥珀酸合成酶之间几乎不存在其他序列同源性。X射线衍射研究的基础上，折叠拓扑结构的合成酶和p21 rasproteins是不同的。然而，与p21 ras蛋白中GTP相互作用的残基存在于合成酶中几乎相同的位置。因此，我们选择研究G15 V突变体，一个磷酸结合环突变体，和R331 L和R331 R，两个突变体的赖氨酸331参与鸟嘌呤环结合。腺苷酸琥珀酸合成酶突变体K331 L和K331 R对GTP的Km值分别比野生型酶增加了27倍和20倍，而IMP的Km值没有任何显著变化。因为两种突变对GTP的Km值的影响相似，而kcat和二级结构基本上没有变化，这表明Lys 331位于腺苷酸琥珀酸合成酶的GTP结合位点，其末端N-xi对GTP的结合并不重要。因此，Lys 331可能通过其线性侧链与GTP的鸟嘌呤碱基的芳环之间的疏水相互作用与GTP相互作用。此外，使用圆二色性（CD）光谱法，NMR光谱法和荧光分光光度法进行G15 V突变体的结构表征。CD光谱数据表明，G15 V突变体的二级结构显着改变GTP和IMP，而野生型酶的二级结构没有改变，然而，这两种酶表现出相似的二级结构，在没有底物。两种酶的NMR光谱在不存在底物的情况下也是相似的。G15 V突变体对IMP的解离常数（Kd）是其Km值的4.8倍，而Km值是野生型酶的1.5倍。从这些研究结果中，可以得出结论，腺苷酸琥珀酸合成酶的磷酸盐结合区域参与GTP和IMP结合诱导的构象变化，并且GTP和LMP结合依赖于酶的活性位点处的其他底物的存在。这些结果表明腺苷酸琥珀酸合成酶的Lys 331在结构和功能上可能与GTP结合蛋白相似。然而，磷酸结合环除了与GTP的γ-磷酸相互作用之外还有其他功能。
Adenylosuccinate synthetase contains amino acid sequences in its GTP-binding domain that are homologous to other G-proteins. This homology includes a glycine-rich phosphate-binding loop, GXXXXGK, and a guanine-specific binding region, (N/T/Q)KXD; however, virtually no other sequence homology exists between other G-proteins and adenylosuccinate synthetase. On the basis of X-ray diffraction studies, the folding topologies of the synthetase and the p21rasproteins are different. Yet, residues that interact with GTP in the p21rasproteins are present in the synthetase in nearly identical positions. We chose therefore to study the G15V mutant, a phosphate-binding loop mutant, and R331L and R331R, two mutants of Lys331that are involved in guanine ring binding. TheKmvalues for GTP of adenylosuccinate synthetase mutants, K331L and K331R, when compared to those of the wild-type enzyme, were 27- and 20-fold increased, respectively, without any significant change in theKmvalues for IMP. Because both mutations affected theKmvalues for GTP similarly, whereas thekcatand secondary structure were essentially unchanged, it is suggested that Lys331is located in the GTP-binding site of adenylosuccinate synthetase and the terminal N-xi of the Lys is not necessarily important in GTP-binding on the enzyme. Therefore, Lys331may interact with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant was carried out using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry. The CD spectral data indicated that the secondary structure of the G15V mutant was significantly altered by GTP and IMP, whereas that of the wild-type enzyme is not changed; however, the two enzymes exhibited similar secondary structures in the absence of substrates. The NMR spectra of both enzymes were also similar in the absence of substrates. The dissociation constant (Kd) for IMP of the G15V mutant was 4.8-fold larger than itsKmvalue which was 1.5-fold increased compared to the wildtype enzyme. From these findings, it was concluded that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and LMP binding depend on the presence of the other substrate at the active site of the enzyme. These results suggest that the Lys331of adenylosuccinate synthetase may play similar roles in the function and structure to that of GTP-binding proteins. However, the phosphate-binding loop has additional functions besides the interaction with the gamma-phosphate of GTP.