Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea.
Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea.
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天冬氨酸转氨甲酰酶锌结构域几何形状的定点改变：选择性改变异向配体、等电点和尿素稳定性的调节。
DOI：
10.1021/bi00067a002
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Wild,JR
中科院分区：
文献类型：
--
作者：
Strang,CJ;Wales,ME;Brown,DM;Wild,JR
1992年12月2日收到的修订手稿摘要：通过在锌结构域中催化链和调节链接触区域的特定氨基酸插入或替换，探索了天冬氨酸转氨酶调节链中变构功能所必需的结构方面。在两个位置上的氨基酸取代产生的酶保持了与野生型酶相似的最大速度，但在调节核苷三磷酸存在的情况下与天然酶的反应不同。在pH 7时，锌配位氨基酸Cl09转变为组氨酸，EL 19转变为天冬氨酸，CTP和UTP表现出协同抑制作用，而CTP没有抑制作用。在pH 8.3时，天然酶中存在较高比例的T态构象，突变株表现出与其相似的动力学行为。C109H仍然是一种不受CTP抑制但仍被CTP+UTP抑制的酶。CTP和CTP+UTP对EL 19D均有抑制作用。ATP对突变体的激活作用随pH或以磷酸盐为缓冲成分而变化。在磷酸盐中，C109H被ATP激活，而在pH为7或8.3的TP中，其被ATP激活的作用减弱或消失。在pH为7的磷酸盐溶液或在pH为8.3的TP溶液中，EL 19D被ATP激活，但在pH为7的TP溶液中不被激活。未连接突变体酶的S值和希尔系数与野生型酶相似。虽然这两个突变都不会改变全酶的净电荷，但如果有磷酸盐存在，就会观察到突变体等电点的差异。这一结果表明，天冬氨酸转氨甲酰基酶的等电点是构象依赖的，突变株以改变的构象存在。此外，两种突变型全酶在4M尿素中的稳定性均显著低于野生型。在Tris缓冲液中，C109H在pH 8.25时较稳定；在磷酸盐缓冲液中，EL 19D在pH 7时较稳定。讨论了这些突变对活性中心化学和几何构型的潜在影响。大肠埃希菌的天冬氨酸氨基转移酶(ATCase，氨基甲酰-磷酸：L-天冬氨酸氨基甲酰转移酶，EC2.1。3.2)是一种变构酶，长期研究其底物诱导的协作性和异向性调节分子对动力学性质的影响[有关综述，请参阅Stevens等人(1991年)、Lipscomb(1991年)、Perutz(1990年)、Allewell()、Kantrowitz和Lipscomb(1988)、Schachman(1988)、Kantrowitz等人(1980a，b)。这种酶
Revised Manuscript Received December 2, 1992 abstract: Structural aspects requisite for allosteric function in the regulatory chain of aspartate transcarbamoylase were explored by site-specific amino acid insertion or substitution within the zinc domain in the region of contact between the catalytic and regulatory chains. Amino acid substitution at two positions yielded enzymes which retained a maximum velocity similar to that of the wild-type enzyme but responded differently from the native enzyme in the presence of regulatory nucleoside triphosphates. A change of zinc coordinate amino acid Cl09 to histidine and a change of El 19 to aspartic acid resulted in enzymes which demonstrated synergistic inhibition by CTP and UTP but not inhibition by CTP in either phosphate buffer or a morpholino-based tripartate (TP) buffer at pH 7. At pH 8.3, where there is a higher proportion of T-state conformers in the native enzyme, the mutants diverged from their similar kinetic behavior. C109H remained an enzyme which was not inhibited by CTP but was still inhibited by CTP+ UTP. El 19D was inhibited by bothCTP and CTP+ UTP. Activation of the mutants by ATP was found to vary either with pH or with phosphate as a buffer component. C109H was activated by ATP in phosphate, while in TP at either pH 7 or 8.3 its activation by ATP was diminished or absent. El 19D was activated by ATP in phosphate at pH 7 or in TP at pH 8.3, but not in TP at pH 7. In TP at pH 7, where neither mutant was activated by ATP, the So. s values and Hill coefficients of the unliganded mutant enzymes resembled those of the ATP-liganded wild-type enzyme. While neither mutation would be predicted to alter the net charge of the holoenzyme, differences in the isolectric point of the mutants were observed if phosphate was present. This result suggests that the isoelectric point of aspartate transcarbamoylase is conformationally dependent and that the mutants exist in an altered conformation. In addition, the stabilities of both mutant holoenzymes were reduced substantiallyfrom those of the wild-type enzyme in 4 M urea. C109H was more stable at pH 8.25 in a Tris buffer; El 19D was more stable at pH 7 in the phosphate buffer. Potential effects of these mutations on the active site chemistry and geometry are discussed.Aspartate transcarbamoylase of Escherichia coli (ATCase, carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1. 3.2) is an allosteric enzyme of long-standing investigation for itssubstrate induced cooperativity and the effect of heterotropic regulatory molecules on the kinetic properties [for reviews, see Stevens et al.(1991), Lipscomb (1991), Perutz (1990), Allewell (1989), Kantrowitz and Lipscomb (1988), Schachman (1988), Kantrowitz et al.(1980a, b)]. This enzyme
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天冬氨酸转氨甲酰酶的调节和催化活性的不同亚基。
影响因子：
2.9
作者：
J. Gerhart;H. K. Schachman
通讯作者：
H. K. Schachman
天冬氨酸转氨甲酰酶中不同亚基之间的通讯：抑制剂和激活剂对催化多肽链构象的影响。
DOI：
--
发表时间：
1979
影响因子：
11.1
作者：
P. Hensley;H. K. Schachman
通讯作者：
H. K. Schachman
天冬氨酸转氨甲酰酶与胞苷 5-三磷酸和腺苷 5-三磷酸相互作用的平衡结合研究。
影响因子：
2.9
作者：
S. Matsumoto;G. Hammes
通讯作者：
G. Hammes
天冬氨酸转氨甲酰酶中 CTP 和 ATP 位点的结合和物理分配的不对称性。
影响因子：
4.8
作者：
P. Suter;J. Rosenbusch
通讯作者：
J. Rosenbusch
天冬氨酸转氨甲酰酶中的亚基相互作用。
影响因子：
4.8
作者：
W. W. Chan
通讯作者：
W. W. Chan