RESUMO
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.
Assuntos
Ácido Aspártico/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting , Escherichia coli/enzimologia , Escherichia coli/química , Nuclease do Micrococo/metabolismo , Mutagênese Sítio-Dirigida , Oligonucleotídeos , Precursores de Proteínas/metabolismo , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/química , Relação Estrutura-AtividadeRESUMO
No abstract available.
RESUMO
No abstract available.