Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies.
Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies.
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通过 pH 研究确定鸟氨酸转氨甲酰酶的底物特异性和质子化状态。
DOI：
10.1021/bi00339a007
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Lipscomb,WN
中科院分区：
文献类型：
--
作者：
Kuo,LC;Herzberg,W;Lipscomb,WN
化学系，哈佛大学，剑桥，马萨诸塞州02138接收1984年10月4日摘要：在pH 5.5和10.5之间研究了鸟氨酸转氨甲酰酶催化的反应和L-正缬氨酸对其的抑制。来自大肠杆菌的酶的稳态周转率（A，*，）随着pH增加，并且在pH 9以上达到平台。其随pH的变化符合一个单一的质子化过程，表观pAfa为7.3。pH值对L-鸟氨酸表观米氏常数（ATMapp）的影响表明，这种阳离子形式的二氨基酸不是底物。仅以两性离子鸟氨酸为底物，反应的假一级速率常数（Acat/KMZ）的pH曲线为钟形曲线，其特征在于pATa为6.2和9.1，渐近斜率为±1。两性离子型L-正缬氨酸（一种竞争性抑制剂）的pAf曲线也得到类似的pAys（6.3和9.3）。pAf曲线进一步表明，抑制剂的-氨基必须带电荷才能结合。总之，这些pH曲线提供了足够的信息，表明只有鸟氨酸的次要两性离子种类H2 N（CH 2）3CH（NH3+）COO-有效地结合酶。酶对这种底物形式的选择导致了一种米氏络合物，其中鸟氨酸准备进行亲核攻击。在这样的结合之后，避免了对δ-NH3+基团的去质子化的需要，并且转氨甲酰化在能量上变得更可行。对于酶促反应，提出了考虑pH影响的反应方案。尿素循环中的N酶，鸟氨酸转氨甲酰酶
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138 Received October 4, 1984 abstract: The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5. The steady-state turnover rate (A,*,) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9. Its change with pH conforms to a single protonation process with an apparent pAfa of 7.3. The effect of pH on the apparent Michaelis constant (ATMapp) of L-ornithine suggests that this diamino acid in its cationicform is not the substrate. Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (Acat/KMZ) of the reaction is a bell-shaped curve characterized by pATa’s of 6.2 and 9.1 and asymptotic slopes of±1. Similar pAys (6.3 and 9.3) are obtained for the pAf¡ profile of zwitterionic L-norvaline, a competitive inhibitor. The pAf¡ profile further indicates that the-amino group of the inhibitor must be charged for binding. Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N (CH2) 3CH (NH3+) COO~, binds the enzyme productively. The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack. Following such binding, the need for deprotonation of the< 5-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible. Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction..^^. n enzyme in the urea cycle, ornithine transcarbamoylase