Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis.
Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) 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 Ca2+ 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 (kcat).
Kasche
Volker
V
Institute of Biotechnology II, Technical University Hamburg-Harburg, Hamburg, Germany. kasche@tu-harburg.de
Galunsky
Boris
B
Ignatova
Zoya
Z
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010446:Peptide Fragments; D011498:Protein Precursors; D010399:Penicillin Amidase