Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli.
The penicillin amidase (PA) from Escherichia coli belongs to a group of proteolytically processed bacterial enzymes. The mechanism of the maturation of the single polypeptide proenzyme has been studied for the PA from E. coli using a slowly processing mutant proenzyme. The mutant proenzyme was constructed 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 PA activity as observed in homogenates of wild-type PA-producing E. coli cells were found to be formed during this process. A kinetic analysis of the possible inter- and intramolecular processes involved in the maturation demonstrated that unambiguous evidence for the existence of intramolecular processes can 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 and immobilized mutant proenzyme, based on determinations of mass spectra, N-terminal sequences and active site concentrations. In the system with immobilized proenzyme intermolecular processes are excluded, demonstrating that this bond is hydrolysed by intramolecular autoproteolysis. Based on the known three-dimensional structure of the PA from E. coli the same maturation mechanism should apply for the wild-type proenzyme.
Kasche
V
V
AB Biotechnologie II, TU-Hamburg-Harburg, Denickestrasse 15, D-21071, Hamburg, Germany. kasche@tu-harburg.de
Lummer
K
K
Nurk
A
A
Piotraschke
E
E
Rieks
A
A
Stoeva
S
S
Voelter
W
W
D001665:Binding Sites; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D013058:Mass Spectrometry; D015394:Molecular Structure; D009154:Mutation; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D004800:Enzymes, Immobilized; D010450:Endopeptidases; D010399:Penicillin Amidase