Refolding and assembly of penicillin acylase, an enzyme composed of two polypeptide chains that result from proteolytic activation.
The in vitro folding and assembly of penicillin acylase (EC 3.5.1.11) (PA) to active enzyme has been studied. PA is a large bacterial protein (Mr = 86,000) comprising two peptides, alpha and beta, produced by proteolytic processing and activation of a 92-kDa precursor. Proteins that result from proteolytic processing are characteristically difficult if not impossible to refold. Different factors that affect folding and assembly of PA, including pH, ionic strength, and temperature, have been studied. Yields of 60% can be obtained, based on recovery of enzyme activity, together with another 20% of folded and associated monomer with conformation closely similar to that of the active enzyme but with the active site not formed. Evidence is presented for in vitro assembly proceeding via initial folding of the N-terminal alpha-peptide with subsequent collapse of the transiently folded beta-chain on to the surface of the former. A slow process of rearrangement follows association in vitro. Competition experiments support the proposal that the linker endopeptide in the precursor serves to increase the probability of productive collision between folded alpha- and beta-peptides. The effect of raised temperature is to interfere with the folding of the alpha-peptide, thus preventing proper folding of the precursor. This finding accounts for the basis of the temperature regulation of PA production in vivo.
Lindsay
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CD
Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K.
Pain
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RH
D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009994:Osmolar Concentration; D010399:Penicillin Amidase / Q000737:chemistry*; D010447:Peptide Hydrolases; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D013696:Temperature
D010455:Peptides; D010447:Peptide Hydrolases; D010399:Penicillin Amidase