REMODELING HEXOSE-1-PHOSPHATE URIDYLYLTRANSFERASE - MECHANISM-INSPIRED MUTATION INTO A NEW ENZYME, UDP-HEXOSE SYNTHASE
REMODELING HEXOSE-1-PHOSPHATE URIDYLYLTRANSFERASE - MECHANISM-INSPIRED MUTATION INTO A NEW ENZYME, UDP-HEXOSE SYNTHASE
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DOI：
10.1021/bi00499a003
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发表时间：
1990-11-27
期刊：
影响因子：
2.9
通讯作者：
FREY, PA
中科院分区：
文献类型：
--
作者：
KIM, JM;RUZICKA, F;FREY, PA
己糖-1-磷酸尿苷酰转移酶催化UDP-半乳糖和葡萄糖-1-P与UDP-葡萄糖和半乳糖-1-P通过共价中间体（E-UMP）的双置换机制相互转化，其中UMP与活性位点H164或H166处的两个组氨酸残基之一键合。为了确定组氨酸是亲核催化剂，我们准备了两个特定的突变体的酶从大肠杆菌，H164 G和H166 G，其中每个咪唑环和亚甲基碳的一个组氨酸被删除。为了确定突变体中缺失的咪唑的功能是否可以通过尿苷5“-（磷酸咪唑酯）（UMP-Im）中的咪唑环来实现，我们检测了突变体蛋白在UMP-Im与葡萄糖-1-P反应以形成UDP-葡萄糖和咪唑中的催化活性。突变体H166 G催化该反应，以及逆反应，通过一个连续的动力学机制，涉及三元复合物作为中间体。该突变酶还接受半乳糖-1-P作为底物以形成UDP-半乳糖。己糖-1-P尿苷酰转移酶不催化这些反应，并且H166 G不催化野生型反应。突变酶的底物Km值与己糖-1-P尿苷酰转移酶的Km值相似。在UDP-葡萄糖形成方向上的Kcat值为1.31 ±。0.01 s-1，而己糖-1-P尿苷酰转移酶为350 s-1，并且在相反方向上kcat为4.8 ± 1。0.4 s-1，而野生型酶为960 s-1。因此，动力学的“价格”，这是为消除共价键和亚甲基基团连接的酶和亲核咪唑在母体酶是一个约250倍的营业额下降。突变体H164 G没有检测到催化活性。我们认为H166的咪唑环是己糖-1-P尿苷酰转移酶的亲核催化基团。突变蛋白H166 G是一种新的酶，它通过不同于其亲本酶的动力学机制催化新的反应。我们建议将这种新酶命名为UDP-己糖合酶。
Hexose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-galactose and glucose-1-P with UDP-glucose and galactose-1-P by a double-displacement mechanism through a covalent intermediate (E-UMP), in which UMP is bonded to one of two histidine residues at the active site, H164 or H166. To identify which histidine is the nucleophilic catalyst, we prepared two specific mutants of the enzyme from Escherichia coli, H164G and H166G, in each of which the imidazole ring and methylene carbon of one histidine are deleted. To determine whether the function of the deleted imidazole in the mutants could be carried out by the imidazole ring in uridine 5''-(phosphoimidazolate) (UMP-Im), we examined the mutant proteins for catalytic activity in the reaction of UMP-Im with glucose-1-P to form UDP-glucose and imidazole. The mutant H166G catalyzes this reaction, as well as the reverse reaction, by a sequential kinetic mechanism involving ternary complexes as intermediates. The mutant enzyme also accepts galactose-1-P as a substrate to form UDP-galactose. Hexose-1-P uridylyltransferase does not catalyze these reactions, and H166G does not catalyze the wild-type reaction. The substrate Km values for the mutant enzyme are similar to those for hexose-1-P uridylyltransferase. The value of Kcat in the direction of UDP-glucose formation is 1.31 .+-. 0.01 s-1 compared with 350 s-1 for hexose-1-P uridylyltransferase, and in the reverse direction kcat is 4.8 .+-. 0.4 s-1, compared with 960 s-1 for the wild-type enzyme. Thus, the kinetic "price" that is paid for removing the covalent bond and a methylene group linking the enzyme and the nucleophilic imidazole in the parent enzyme is an approximately 250-fold decrease in turnover number. The mutant H164G has no detectable catalytic activites. We conclude that the imidazole ring of H166 is the nucleophilic catalytic group of hexose-1-P uridylyltransferase. The mutant protein H166G is a new enzyme that catalyzes a new reaction by a kinetic mechanism different from that of its parent enzyme. We suggest the name UDP-hexose synthase for this new enzyme.