Identification of potential active-site residues in the Escherichia coli leader peptidase.
Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane. Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown. This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W. (1981) J. Biol. Chem. 256, 3593-3597). Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups. We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Replacement of each histidine residue with an alanyl residue was without effect on catalysis. Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract. However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay. In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase. Replacement of this residue with alanine results in a very unstable leader peptidase protein. This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.
Sung
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Department of Chemistry, Ohio State University, Columbus 43210.
Dalbey
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D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases
D036022:Capsid Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; C025668:coat protein, Bacteriophage M13; D001224:Aspartic Acid; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D000409:Alanine