Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure.
Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure.
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DOI：
10.1016/j.bbrc.2011.06.166
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发表时间：
2011-08-05
影响因子：
3.1
通讯作者：
Anderson, Mary E.
中科院分区：
文献类型：
--
作者：
Brown, Teresa R.;Drummond, Michael L.;Barelier, Sarah;Crutchfield, Amanda S.;Dinescu, Adriana;Slavens, Kerri D.;Cundari, Thomas R.;Anderson, Mary E.
人谷胱甘肽合成酶（hGS）催化谷胱甘肽（GSH）生物合成的第二个依赖atp的步骤，并与γ-谷氨酰底物负合作。hGS活性位点由3个高度保守的催化环组成，特别是富丙氨酸a环。本文报道了Asp458突变影响的实验和计算研究，从而定义了该a环残基对hGS结构、活性、负协同性和稳定性的作用。利用定点诱变技术构建了几个Asp458 hGS突变体（D458A、D458N、D458R），并测定了它们的活性（分别占野生型hGS的10%、15%和7%）。测定了三种底物（甘氨酸、GAB、ATP）的Michaelis-Menten常数（Km）：甘氨酸Km增加30 ~ 115倍，GAB Km减少8 ~ 17倍，ATP Km不变。所有的Asp458突变体都表现出从负合作性到非合作性的变化。通过差示扫描量热法测定，所有突变体都表现出与野生型hGS相似的稳定性。研究结果表明，Asp458对hGS的催化至关重要，并影响hGS的变构。
Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N, D458R) were constructed using site-directed mutagenesis and their activities determined (10, 15 and 7% of wild-type hGS, respectively). The Michaelis-Menten constant (Km) was determined for all three substrates (glycine, GAB, ATP): glycine Km increased by 30 - 115 fold, GAB Km decreased by 8 - 17 fold, and the ATP Km was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.
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影响因子：
5.5
作者：
Hess, Berk;Kutzner, Carsten;Lindahl, Erik
通讯作者：
Lindahl, Erik
DOI：
10.1073/pnas.62.4.1121
发表时间：
1969-01-01
影响因子：
11.1
作者：
LEVITZKI, A;KOSHLAND, DE
通讯作者：
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影响因子：
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作者：
Dinescu, A;Cundari, TR;Anderson, ME
通讯作者：
Anderson, ME
DOI：
10.1016/j.bbrc.2010.08.081
发表时间：
2010-10-01
影响因子：
3.1
作者：
Dinescu A;Brown TR;Barelier S;Cundari TR;Anderson ME
通讯作者：
Anderson ME
影响因子：
2.1
作者：
GASTEIGER, J;MARSILI, M
通讯作者：
MARSILI, M