Kinetic analysis of three activated phenylalanyl intermediates generated by the initiation module PheATE of gramicidin S synthetase

The three-domain initiation module PheATE (GrsA) of Bacillus brevis gramici din S synthetase catalyzes the activation, thiolation and epimerization of L-phenylalanine (L-Phe), the first amino acid incorporated into the decapep tide antibiotic gramicidin S, There are three activated intermediates in th e PheATE catalyzed chemical pathway: L-phenylalanyl-adenosine-5'-monophosph ate 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 c atalysis using rapid chemical quench techniques. Kinetic modeling of the pr ocess of disappearance of the substrate L-Phe, transient appearance and dis appearance 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 micr oscopic rate constants for the three chemical reactions in the initiation m odule PheATE. This study provides the first transient-state kinetic analysi s of a nonribosomal peptide synthetase (NRPS) module.