ACYL-COENZYME-A-ISOPENICILLIN-N-ACYLTRANSFERASE FROM PENICILLIUM-CHRYSOGENUM - EFFECT OF AMINO-ACID SUBSTITUTIONS AT SER(227), SER(230) ANDSER(309) ON PROENZYME CLEAVAGE AND ACTIVITY

Using a high level Escherichia coli expression system for the Penicill ium chrysogenum penDE gene, we have produced acyl-coenzyme A:isopenici llin N acyltransferase (AT) enzymes containing amino acid substitution s at three conserved Ser residues. Chosen for study based on amino aci d sequence homologies to other proteins, Ser(227), Ser(230) and Ser(30 9) were changed to Cys or Ala to assess amino acid side chain involvem ent in proenzyme cleavage and AT enzyme mechanism. Substitutions at Se r(230) had no effect on proenzyme cleavage, acyl-coenzyme A:IPN acyltr ansferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransf erase (AAT) activities. While Ser(227) --> Cys had no effect, Ser(227) --> Ala produced uncleaved proenzyme lacking both AAT and IAT activit ies, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitut ion of Ser(309) --> Cys did not appreciably prevent proenzyme cleavage , IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser (309) --> Ala was cleaved; however, IAT and AAT activities were not ob served. This separation of proenzyme cleavage from IAT and AAT activit ies has not been previously observed, and suggests that Ser(309) is in volved in substrate acylation.