13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase.
13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase.
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13C 同位素效应作为大肠杆菌天冬氨酸转氨甲酰酶动力学机制和变构特性的探针。
DOI：
10.1021/bi00143a029
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Cleland,WW
中科院分区：
文献类型：
--
作者：
Parmentier,LE;O'Leary,MH;Schachman,HK;Cleland,WW
1992年4月21日摘要：在天冬氨酸转氨甲酰酶催化的反应中，测定了氨甲酰磷酸中13 C的动力学同位素效应。对于全酶，在天冬氨酸为零时，该值为1.0217，但在天冬氨酸为无穷大时，该值为1，4.8 mM天冬氨酸消除了一半的同位素效应。这种模式证明了一种有序的动力学机制，氨基甲酰磷酸在天冬氨酸之前加入。在ATP或CTP的存在下观察到相同的参数，表明无论是否存在变构修饰剂，仅存在一种形式的活性酶。这些数据支持变构行为的Monod模型，其中完全活性和非活性酶之间的平衡被底物和修饰剂的选择性结合相互作用扰乱，并且没有具有部分活性的酶形式。分离的催化亚基的酶表现出类似的13 C同位素效应（1.0240在零天冬氨酸，1.0039在无限天冬氨酸，3.8 mM天冬氨酸造成一半的变化从一个值到另一个），但在无限天冬氨酸的有限同位素效应表明，动力学机制现在部分随机。与非常缓慢和结合不良的天冬氨酸类似物半胱氨酸亚磺酸盐，13 C同位素效应为1.039全酶和催化亚基，并没有显着降低高水平的半胱氨酸亚磺酸盐。1.039的值可能接近于化学反应的内在同位素效应，而这种底物的动力学机制现在是完全随机的，因为化学反应比酶释放任何一种反应物慢得多。天冬氨酸转氨甲酰酶（EC 2.1. 3.2，ATCase）1是一种高度调节的酶，其在嘧啶生物合成的第一个关键步骤中催化由L-天冬氨酸和氨甲酰磷酸形成N-氨甲酰-L-天冬氨酸和无机磷酸。ATCase表现出同向和异向相互作用（Gerhart & Pardee，1962; Bethel等人，1968年）。在L-天冬氨酸或氨甲酰磷酸存在下，可观察到正协同作用，其饱和曲线呈S形。这种对底物浓度的S形依赖性通常被认为是底物诱导的构象变化的结果，所述构象变化引起底物浓度的变化。
Revised Manuscript Received April 21, 1992 abstract: 13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase. For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect. This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate. The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers. These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates andmodifiers, and there are no enzyme forms having partial activity. Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random. With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate. The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme.. Aspartate transcarbamylase (EC 2.1. 3.2, ATCase) 1 is a highly regulated enzyme that catalyzes the formation of N-carbamyl-L-aspartate and inorganic phosphate from L-as-partate and carbamyl phosphate in the first committed step of pyrimidine biosynthesis. ATCase exhibits both homotropic and heterotropic interactions (Gerhart & Pardee, 1962; Bethel et al., 1968). Positive cooperativity, in the form of sigmoidal saturation curves, isseen in thepresence of either L-aspartate or carbamyl phosphate. This sigmoidal dependence on sub-strate concentration is generally felt to be the result of a substrate-induced conformational change which causes the
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大肠杆菌天冬氨酸转氨甲酰酶的效应子会不同程度地干扰天冬氨酸结合，而不是 T-R 转换。
DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Hsuanyu,YC;Wedler,FC
通讯作者：
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10.1016/s0021-9258(18)99588-8
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1967-06
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天冬氨酸转氨甲酰酶催化亚基催化的反应机理。
影响因子：
2.9
作者：
Elizabeth Heyde;A. Nagabhushanam;J. F. Morrison
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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：
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作者：
Foote,J;Lauritzen,AM;Lipscomb,WN
通讯作者：
Lipscomb,WN
测定重原子同位素对酶催化反应的影响。
影响因子：
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作者：
M. O'Leary
通讯作者：
M. O'Leary