Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate.

摘要: Peptide amidase (Pam), a hydrolytic enzyme that belongs to the signature (AS) family, selectively catalyzes hydrolysis of C-terminal amide bond (CO-NH(2)) peptides. The recent availability X-ray structures Pam, fatty acid hydrolase, and malonamidase E2 has led proposal novel Ser-Ser-Lys catalytic triad mechanism for by AS enzymes. molecular dynamics (MD) simulations using CHARMM force field were performed explore Pam. 1.8 A crystal structure Pam in complex with analogue chymostatin was chosen initial coordinates MD simulations. five systems investigated are as follows: (i) enzyme.substrate Lys123-NH(2), (ii) Lys123-NH(3)(+), (iii) Lys123-NH(3)(+) Ser226-O(-), (iv) enzyme.transition state, (v) enzyme.tetrahedral intermediate. Our data support presence hydrogen bonding network among residues, Ser226, Ser202, Lys123, where Ser226 acts nucleophile Ser202 bridges Lys123. simulation supports role crystallographic waters, Wat1 Wat2. In all have been studied, backbone nitrogens Asp224 Thr223 create an oxyanion hole terminal oxygen substrate, stabilize tetrahedral results from both our computational investigation previously published experimental pH profile two mechanisms. is relevant at lower pH, Lys123-NH(3)(+)-Ser202 dyad provides structural residue which turn carries out nucleophilic attack substrate carbonyl concert Wat1-mediated deprotonation stabilization transition state hole. operating higher Lys123-NH(2)-Ser202 general base assist addition carbonyl. intermediate show Lys123 possible candidates protonation leaving group, NH(2), form acyl-enzyme