The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.
The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.
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苏氨酸 55 对天冬氨酸转氨甲酰酶催化的贡献。
DOI：
10.1021/bi00143a032
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Schachman,HK
中科院分区：
文献类型：
--
作者：
Waldrop,GL;Turnbull,JL;Parmentier,LE;Lee,S;O'Leary,MH;Cleland,WW;Schachman,HK
1992年4月20日摘要：重原子同位素效应和稳态动力学参数进行了测量的催化三聚体的活性位点突变体的天冬氨酸氨基甲酸转移酶，T55 A，以评估的作用，苏氨酸55在催化。与野生型酶相比，氨基甲酰磷酸与T55 A突变体的结合减少了2个数量级，而对琥珀酸和琥珀酸的亲和力没有明显改变。这表明Thr 55在CbmP的结合中起重要作用。如果，如先前所建议的，Thr 55协助CbmP的羰基的极化，T55 A突变体的碳同位素效应相对于野生型酶所观察到的应该增加。然而，相反的是看到，表明Thr 55不参与稳定过渡态的含氧阴离子。一系列13 C和15 N同位素效应的定量分析表明，T55 A三聚体催化反应的速率决定步骤可能是米氏复合物形成后蛋白质的构象变化。因此，Thr 55可以促进酶的构象变化，这是催化的先决条件。具有T55 A三聚体的二元和米氏复合物中改变的活性位点环境反映在log V、log（V/K）asp和pK {琥珀酸酯的pH曲线中，其显示相对于野生型酶参与天冬氨酸结合和催化的电离残基的pK值的位移。双底物类似物A-（膦酰基乙酰基）-L-天冬氨酸对T55 A三聚体的抑制作用与天冬氨酸呈竞争性，其初速度模式为交叉型。这些结果表明，苏氨酸突变为丙氨酸改变了动力学机制，从稳态有序到快速平衡随机。根据连接到双底物类似物N-（膦酰基乙酰基1）-1-天冬氨酸（PALA）上的天冬氨酸转氨甲酰酶（ATCase）1的晶体结构，已经推断活性位点残基Thr 55与氨甲酰天冬氨酸（CbmP）的羰基氧和磷酸部分相互作用（Krause等人，1987年）。这一假设导致Lipscomb及其同事推测Thr 55可能通过帮助使CbmP的羰基极化而参与催化（Gouaux等人，1987年）。Xu和Kantrowitz（1989）在注意到Thr 55被丙氨酸取代导致最大活性相对于野生型酶降低5倍后同意该提议。在先前的论文（Waldrop等人，1992年a），它表明，应用重原子同位素效应可以提供有价值的信息，在确定如何一个活性位点μ-
Revised Manuscript Received April 20, 1992 abstract: Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzymewhereas the affinities for aspartateand succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent toformation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K) asp, and pK {succinate, which show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme. Inhibition of the activity of T55A trimer bythe bisubstrate analogue A-(phosphonoacetyl)-L-aspartate was competitive with respect to aspartate, and the initial velocity pattern was of the intersecting type. These results indicate that the threonine toalanine mutation alters the kinetic mechanism from steady-state ordered to rapid-equilibrium random.^) n the basis of the crystal structure of aspartate trans-carbamoylase (ATCase) 1 ligated to the bisubstrate analogue N-(phosphonoacety 1)-l-aspartate (PALA), it has been inferred that the active site residue, Thr 55, interacts with both the carbonyl oxygen and phosphate moieties of carbamoyl phos-phate (CbmP)(Krause et al., 1987). This hypothesis led Lipscomb and co-workers to speculate thatThr 55 may participate in catalysis by assisting in polarizing the carbonyl group of CbmP (Gouaux et al., 1987). Xu and Kantrowitz (1989) concurred with this proposal after noting that the replacement of Thr 55 by alanine resulted in a reduction of the maximal activity by a factor of 5 relative to the wild-type enzyme. In the previous paper (Waldrop et al., 1992a), it was shown that the application of heavy-atom isotope effects could provide valuable information in determining how an active site mu-
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大肠杆菌天冬氨酸氨基甲酰转移酶的催化机制：分子模型研究。
DOI：
10.1016/0006-291x(87)91497-5
发表时间：
1987
影响因子：
3.1
作者：
Gouaux,JE;Krause,KL;Lipscomb,WN
通讯作者：
Lipscomb,WN
DOI：
10.1016/s0021-9258(18)99588-8
发表时间：
1967-06
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. Gerhart;H. Holoubek
通讯作者：
J. Gerhart;H. Holoubek
天冬氨酸转氨甲酰酶的底物特异性。
DOI：
--
发表时间：
1985
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Foote,J;Lauritzen,AM;Lipscomb,WN
通讯作者：
Lipscomb,WN
[5]天冬氨酸转氨甲酰酶（大肠杆菌）：亚基的制备
DOI：
--
发表时间：
1978
期刊：
影响因子：
--
作者：
Ying R. Yang;M. Kirschner;H. K. Schachman
通讯作者：
H. K. Schachman
天冬氨酸转氨甲酰酶的位点特异性诱变。
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Robey,EA;Schachman,HK
通讯作者：
Schachman,HK