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

The variation of kinetic parameters of penicillin V acylase from Streptomyc es lavendulae with pH was used to gain information about the chemical mecha nism of the hydrolysis of penicillin V catalyzed by this enzyme. The pH-dep endence of V-max showed that a group with a pK value of 6.45 (pK(1)) must b e unprotonated for activity. The pH-dependence of V-max/K-m showed that a g roup with a pK value of 7.1 (pK(1)) must be unprotonated and a group with a pK of 10.83 (pK(2)) must be protonated for activity. The lower pK value co rresponded to a group in the enzyme involved in catalysis and whose protona tion state also affects binding. The higher pK value was only involved in b inding. Results from chemical modification studies showed the importance of serine residues in the catalytic mechanism of the enzyme and pointed to th e identity of the groups responsible for pK(1) and pK(2) as the alpha -amin o nitrogen of the N-terminal residue and a lysine residue, respectively. (C ) 2001 Elsevier Science BN. All rights reserved.