Luo L, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2001 May 8;40(18):5329-37. doi: 10.1021/bi015518+.
The three-domain initiation module PheATE (GrsA) of Bacillus brevis gramicidin S synthetase catalyzes the activation, thiolation and epimerization of L-phenylalanine (L-Phe), the first amino acid incorporated into the decapeptide antibiotic gramicidin S. There are three activated intermediates in the PheATE catalyzed chemical pathway: L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), L-Phe-S-4'-phosphopantetheine(Ppant)- and D-Phe-S-4'-Ppant-acyl enzyme. In this study, we examined PheATE in single-turnover catalysis using rapid chemical quench techniques. Kinetic modeling of the process of disappearance of the substrate L-Phe, transient appearance and disappearance of L-Phe-AMP and the ad seriatim formation and equilibration of the L- and D-Phe-S-Ppant-acyl enzyme adducts allowed evaluation of the microscopic rate constants for the three chemical reactions in the initiation module PheATE. This study provides the first transient-state kinetic analysis of a nonribosomal peptide synthetase (NRPS) module.
短短芽孢杆菌短杆菌肽S合成酶的三结构域起始模块PheATE（GrsA）催化L-苯丙氨酸（L-Phe）的活化、硫醇化和差向异构化，L-Phe是掺入十肽抗生素短杆菌肽S的第一个氨基酸。在PheATE催化的化学途径中有三种活化中间体：L-苯丙氨酰-腺苷-5'-单磷酸二酯（L-Phe-AMP）、L-Phe-S-4'-磷酸泛酰巯基乙胺（Ppant）和D-Phe-S-4'-Ppant-酰基酶。在本研究中，我们使用快速化学淬灭技术研究了单轮催化中的PheATE。对底物L-Phe消失过程、L-Phe-AMP的瞬时出现和消失以及L-和D-Phe-S-Ppant-酰基酶加合物的依次形成和平衡进行动力学建模，从而可以评估起始模块PheATE中三个化学反应的微观速率常数。本研究首次对非核糖体肽合成酶（NRPS）模块进行了瞬态动力学分析。
