Asp 280 residue is important in the activity of the Escherichia coli leader peptidase.
Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.
Biochemistry Department, College of Medicine, Kwandong University, Korea.
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D001224:Aspartic Acid / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D015153:Blotting, Western; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D008565:Membrane Proteins; D008836:Micrococcal Nuclease / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D011498:Protein Precursors / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D008565:Membrane Proteins; D009841:Oligonucleotides; D011498:Protein Precursors; C092767:pro-OmpA nuclease A; D001224:Aspartic Acid; D008836:Micrococcal Nuclease; D012697:Serine Endopeptidases; C026515:type I signal peptidase