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 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.
Luo
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
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D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D049148:Aminoacylation; D001051:Apoenzymes / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D002250:Carbon Radioisotopes; D002384:Catalysis; D006096:Gramicidin / Q000096:biosynthesis*; D006639:Histidine / Q000235:genetics; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D010649:Phenylalanine / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics
D001051:Apoenzymes; D002250:Carbon Radioisotopes; D020035:Holoenzymes; C042022:phenylalanyl-AMP anhydride; D006096:Gramicidin; D000249:Adenosine Monophosphate; D010649:Phenylalanine; D010204:Pantetheine; D006639:Histidine; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); C003129:4'-phosphopantetheine; D000409:Alanine