On the catalytic mechanism of prokaryotic leader peptidase 1.
The catalytic mechanism of leader peptidase 1 (LP1) of the bacterium Escherichia coli has been investigated by a combination of site-directed mutagenesis, assays of enzyme activity in vivo utilizing a strain of E. coli which has a conditional defect in LP1 activity, and gene cloning. The biological activity of mutant forms of E. coli LP1 demonstrates that this enzyme belongs to a novel class of proteinases. The possibility that LP1 may be an aspartyl proteinase has been excluded on the basis of primary sequence comparison and mutagenesis. Assignment of LP1 to one of the other three recognized classes of proteinases (metalloproteinases, thiol proteinases and the classical serine proteinases) can also be excluded, as it is clearly demonstrated that none of the histidine or cysteine residues within LP1 are required for catalytic activity. The Pseudomonas fluorescens lep gene has been cloned and sequenced and the corresponding amino acid sequence compared with that of E. coli LP1. The E. coli LP1 and P. fluorescens LP1 primary sequences are 50% identical after insertion of gaps. The P. fluorescens LP1 has 39 fewer amino acids, a calculated molecular mass of 31903 Da and functions effectively in vivo in E. coli. None of the cysteine residues and only one of the histidine residues which are present in E. coli LP1 are conserved in sequence position in the P. fluorescens LP1 enzyme. The possibility that LP1 is a novel type of serine proteinase is discussed.
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SmithKline Beecham Pharmaceuticals, Betchworth, Surrey, U.K.
Munn
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JG
Allsop
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D005798:Genes, Bacterial; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011551:Pseudomonas fluorescens / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases
D008565:Membrane Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase