角色的两个保守氨基酸残基的活性部位galactose-1-phosphate uridylyltransferase:一个重要的丝氨酸和不必要的半胱氨酸。
文章的细节
-
引用
复制到剪贴板 -
凌Geeganage年代,大众,弗雷PA
角色的两个保守氨基酸残基的活性部位galactose-1-phosphate uridylyltransferase:一个重要的丝氨酸和不必要的半胱氨酸。
生物化学。2000年5月9日,39 (18):5397 - 404。
- PubMed ID
-
10820011 (在PubMed]
- 文摘
-
Galactose-1-phosphate uridylyltransferase(高尔特)催化的可逆转变尿苷5 '二磷酸葡萄糖(UDPGlc)和Galactose-1-phosphate尿苷5 '磷酸氢盐半乳糖(UDPGal)和glucose-1-phosphate通过双驱替机理的中间形成共价uridylyl-enzyme (UMP-enzyme)。人民运动联盟之间形成共价键是phosphoramidate一部分和他的166 N高尔特(ε)(2),在他166年的N (delta1)保留一个质子在催化循环。半胱氨酸在大肠杆菌160年和161年Ser高尔特从事氢键的外围磷酰氧原子衬底晶体UMP-enzyme和水晶复杂的H166G-GalT UDPGlc [, j·E。弗雷,p。,Rayment i(1996)生物化学35岁,11560 - 11569;Thoden, j·B。,拉·f·J。弗雷,p。Rayment,我。霍尔顿,h . m .(1997)生物化学36岁,1212 - 1222年)。定点诱变、热力学、动力学瞬态和稳态动力学研究已经进行调查160年半胱氨酸的角色在催化和Ser 161。没有半胱氨酸的硫醇基160年变异C160S C160A并没有严重改变酶活性。然而,变体S161A显示7000倍比野生型高尔特活动更少。 The low activity of S161A was directly related to impaired uridylylation rate constant (3.7 x 10(-)(2) s(-)(1)) and de-uridylylation rate constant (0.5 x 10(-)(2) s(-)(1)) resulting from a higher kinetic barrier for uridylyl-group transfer by the variant S161A as compared with the wild-type GalT. Equilibrium uridylylation studies showed that neither Cys 160 nor Ser 161 was involved in stabilizing the uridylyl-enzyme intermediate. The results lead to the conclusion that the conserved Cys 160 does not play a critical role in catalysis. Ser 161 is most likely involved in donating a hydrogen bond to the beta-phosphoryl group of a substrate, thereby providing proper orientation for nucleophilic catalysis.