Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime.
Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime.
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天然和突变重组酵母乳清酸磷酸核糖基转移酶的克隆、过量生产和纯化，以及磁化反转转移证明所提出的氧代碳正离子中间体不具有动力学寿命。
DOI：
10.1006/abbi.1998.0971
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发表时间：
1999
影响因子：
3.9
通讯作者：
McClard,RW
中科院分区：
文献类型：
--
作者：
Witte,JF;Tsou,R;McClard,RW
来自酿酒酵母的乳清酸磷酸核糖基转移酶基因已亚克隆到大肠杆菌过表达载体中，并已大量生产该酶，从而将纯化简化为一步。我们能够重复已发表的 (J. Victor, L. B. Greenberg, and D. L. SloanJ. Biol. Chem.254, 2647–2655, 1979).32PPi/5-磷酸核糖 1-α-二磷酸交换实验，并且还可以通过磁化反转转移 NMR 实验证明交换。然而，当用 OMP 脱羧酶消除污染的乳清苷 5'-单磷酸 (OMP) 时，任何磁化转移的证据都消失了。因此，得出的结论是，乒乓机制是不可操作的，并且先前提出的沿 OMP 途径的氧代碳正离子中间体在该酶的催化循环中持续的时间不够长，无法被 NMR 交换实验识别。
The gene for orotate phosphoribosyltransferase fromSaccharomyces cerevisiaehas been subcloned into anEscherichia colioverexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step. We were able to repeat the published (J. Victor, L. B. Greenberg, and D. L. SloanJ. Biol. Chem.254, 2647–2655, 1979).32PPi/5-phosphorylribose 1-α-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well. However, when contaminating orotidine 5′-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished. Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments.