Open AccessFragments of pro‐peptide activate mature penicillin amidase of Alcaligenes faecalis
Author(s) -
Kasche Volker,
Galunsky Boris,
Ignatova Zoya
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03871.x
Subject(s) - alcaligenes faecalis , alcaligenes , penicillin , amidase , chemistry , microbiology and biotechnology , antibacterial peptide , peptide , bacteria , antibiotics , biochemistry , biology , enzyme , antibacterial activity , genetics , pseudomonas
Penicillin amidase from Alcaligenes faecalis is a recently identified N‐terminal nucleophile hydrolase, which possesses the highest specificity constant ( k cat / K m ) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca 2+ ion, via a complex post‐translational autocatalytic processing with a multi‐step excision of a small internal pro‐peptide. The function of the pro‐region is so far unknown. In vitro addition of chemically synthesized fragments of the pro‐peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3‐fold. Mutations were used to block various steps in the proteolytic processing of the pro‐peptide to obtain stable mutants with covalently attached fragments of the pro‐region to their A‐chains. These extensions of the A‐chain raised the activity up to 2.3‐fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number ( k cat ).