Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli

The penicillin amidase (PA) from Escherichia coli belongs to a group of pro teolytically processed bacterial enzymes. The mechanism of the maturation o f the single polypeptide proenzyme has been studied for the PA from E. coli using a slowly processing mutant proenzyme; The mutant proenzyme was const ructed by replacing Thr with Gly in the Thr(263)-Ser(264) bond that must be hydrolysed in active PA. The mutant proenzyme was purified by biospecific affinity chromatography using an immobilized monoclonal antibody against PA . The maturation of the free and covalently immobilized purified proenzyme was studied in vitro. For the free proenzyme the same products with PB acti vity as observed in homogenates of wild-type PA-producing E. coli cells wer e found to be formed during this process. A kinetic analysis of the possibl e inter and intramolecular processes involved in the maturation demonstrate d that unambiguous evidence for the existence of intramolecular processes c an only be obtained in systems where intermolecular processes are excluded. The Gly(263)-Ser(264) bond was found to be hydrolysed first in the free an d immobilized mutant proenzyme, based on determinations of mass spectra, N- terminal sequences and active site concentrations. In the system with immob ilized proenzyme intermolecular processes are excluded, demonstrating that this bond is hydrolysed by intramolecular autoproteolysis. Based on the kno wn three-dimensional structure of the PA from E. coli the same maturation m echanism should apply for the wild-type proenzyme. (C) 1999 Elsevier Scienc e B.V. All rights reserved.