Holme E, Lindstedt S
Biochim Biophys Acta. 1982 Jun 4;704(2):278-83. doi: 10.1016/0167-4838(82)90157-1.
The uncoupling of 2-oxoglutarate decarboxylation from hydroxylation in the reaction catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) in the presence of 5-fluorouracil has been studied. In the complete reaction no external reductant is formally needed. The uncoupled reaction is almost negligible in the absence of ascorbate and the optimal ascorbate concentration is 5-times higher than in the presence of a hydroxylatable substrate. This indicates that ascorbate acts as the external reductant that is formally needed in the catalytic cycle. The complete reaction follows the steady-state kinetics of an ordered ter reactant mechanism where 2-oxoglutarate and thymine have to be bound to the enzyme before oxygen (E. Holme (1975) Biochemistry 14, 4999-5003). The uncoupled reaction follows the same kinetic pattern as the complete reaction, and in accordance with this no decarboxylation of 2-oxoglutarate occurs in the absence of a substrate analogue even at elevated oxygen tension. There is a good agreement between Kia values for 2-oxoglutarate of the two reactions, but there is at least a 6-fold increase in KO2 where a minimum value of 25% O2 in the gas phase was found for the partial reaction. The high KO2 found means that the reaction rate could increase considerably at elevated oxygen tension.
在5-氟尿嘧啶存在的情况下，对胸腺嘧啶7-羟化酶（胸腺嘧啶，2-氧代戊二酸：氧氧化还原酶（7-羟化），EC 1.14.11.6）催化的反应中2-氧代戊二酸脱羧与羟基化的解偶联进行了研究。在完整反应中，形式上不需要外部还原剂。在没有抗坏血酸的情况下，解偶联反应几乎可以忽略不计，并且最佳抗坏血酸浓度比存在可羟基化底物时高5倍。这表明抗坏血酸充当催化循环中形式上所需的外部还原剂。完整反应遵循有序三反应物机制的稳态动力学，其中在氧气结合到酶之前，2-氧代戊二酸和胸腺嘧啶必须先结合到酶上（E. Holme（1975年），《生物化学》14，4999-5003）。解偶联反应遵循与完整反应相同的动力学模式，据此，即使在氧气张力升高的情况下，在没有底物类似物的情况下，2-氧代戊二酸也不会发生脱羧反应。两个反应的2-氧代戊二酸的Kia值之间有很好的一致性，但在部分反应中，气相中氧气的最小值为25%时，KO2至少增加了6倍。发现的高KO2意味着在氧气张力升高时反应速率可能会大幅增加。
