Characterization of thimet- and neurolysin-like activities in Escherichia coli M 3 A peptidases and description of a specific substrate.
M 3 A oligopeptidases from Escherichia coli, with hydrolytic properties similar to Zn-dependent mammalian thimet oligopeptidase (EP 24.15) and neurolysin (EP 24.16), were studied aiming at identification of comparative enzyme and substrate specificity, hydrolytic products, and susceptibility to inhibitors. Fluorescent peptides, neurotensin (NT) and bradykinin (BK), were used as substrates for bacterial lysates. Bacterial enzymes were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile, but not by leupeptin, PMSF, E-64, and Z-Pro-Prolinal, using internally quenched Abz-GFSPFRQ-EDDnp as substrate. The molecular mass of the bacterial oligopeptidase activity (77--78 kDa) was determined by gel filtration, and the effect of inhibitors, including captopril, suggested that it results from a combination of oligopeptidase A (OpdA) and peptidyl dipeptidase Dcp (77.1 and 77.5 kDa, respectively). Recombinant OpdA cloned from the same E. coli strain entirely reproduced the primary cleavage of fluorescent peptides, NT and BK, by the bacterial lysate. Genes encoding these M 3 A enzymes were those recognized in E. coli genome, bearing identity at the amino acid level (25--31%) with mammalian Zn-dependent oligopeptidases. We also describe a substrate, Abz-GFSPFRQ-EDDnp, that differentiates bacterial and mammalian oligopeptidases.
Paschoalin
Thaysa
T
Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo (UNIFESP), Rua Botucatu, 862 São Paulo, SP 04023-062, Brazil.
Carmona
Adriana K
AK
Oliveira
Vitor
V
Juliano
Luiz
L
Travassos
Luiz R
LR
D000595:Amino Acid Sequence; D001920:Bradykinin / Q000737:chemistry*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009496:Neurotensin / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009496:Neurotensin; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin; D001920:Bradykinin