Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes.
Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes.
复制标题
二氢新蝶呤醛缩酶的机制。
DOI：
10.1111/j.1742-4658.2007.05761.x
复制
发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Yan,Honggao
中科院分区：
文献类型：
--
作者：
Wang,Yi;Li,Yue;Wu,Yan;Yan,Honggao
二氢新蝶呤醛缩酶（DHNA）催化7，8-二氢-d-新蝶呤（DHNP）裂解形成6-羟甲基-7，8-二氢蝶呤（HP）和乙醇醛，并催化DHNP差向异构化形成7，8-二氢-1-单蝶呤（DHMP）。通过对D2 O溶剂中的反应产物进行NMR分析，研究了差向异构化反应是否使用了与羟醛缩合反应相同的反应中间体或DHNP的C2′的去质子化和再质子化。新形成的DHMP没有观察到C2′的氘化。该结果强烈地表明差向异构化反应使用与羟醛缩合反应相同的反应中间体。与早期的观察相反，DHNA催化的反应是可逆的，这也支持差向异构化反应的非立体特异性逆向醛醇/醛醇机理。用平衡结合和瞬时动力学方法研究了金黄色葡萄球菌DHNA（SaDHNA）和大肠杆菌DHNA（EcDHNA）的结合和催化性质。一套完整的动力学常数的羟醛和差向异构化反应，根据一个统一的动力学机制，确定为SaDHNA和EcDHNA。结果表明，这两种酶具有显著不同的结合和催化性质，这与它们之间的显著序列差异是一致的。
Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8‐dihydro‐d‐neopterin (DHNP) to form 6‐hydroxymethyl‐7,8‐dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8‐dihydro‐l‐monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2′ of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2′ was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA‐catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from bothStaphylococcus aureus(SaDHNA) andEscherichia coli(EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.