Chemical mechanism of penicillin V acylase from Streptomyces lavendulae: pH-dependence of kinetic parameters

Abstract

The variation of kinetic parameters of penicillin V acylase from Streptomyces lavendulae with pH was used to gain information about the chemical mechanism of the hydrolysis of penicillin V catalyzed by this enzyme. The pH-dependence of V max showed that a group with a p K value of 6.45 (p K 1) must be unprotonated for activity. The pH-dependence of V max/ K m showed that a group with a p K value of 7.1 (p K 1) must be unprotonated and a group with a p K of 10.83 (p K 2) must be protonated for activity. The lower p K value corresponded to a group in the enzyme involved in catalysis and whose protonation state also affects binding. The higher p K value was only involved in binding. Results from chemical modification studies showed the importance of serine residues in the catalytic mechanism of the enzyme and pointed to the identity of the groups responsible for p K 1 and p K 2 as the α-amino nitrogen of the N-terminal residue and a lysine residue, respectively.