Tripeptide-specific aminopeptidase from Escherichia coli AJ005

Publisher Website
Google Scholar
Abstract
A tripeptidase, TP, from the ribosome-free fraction of E. coli AJ005, a peptidase-deficient mutant of strain K-12, was obtained using gel electrophoresis and chromatography on DEAE-Sephadex A-50, hydroxylapatite and Sephadex G-200. Characterization studies on tripeptidase TP, freed of other detectable peptidases, indicate that this enzyme is capable of cleaving an amino-terminal leucine, lysine, methionine or phenylalanine residue from certain tripeptides. Only 1 band of activity toward several tripeptides (and no activity toward dipeptides) was detected following gel electrophoresis of this preparation. Tripeptidase TP, the only strain AJ005 peptidase known to attack trilysine, was inactive toward all dipeptides, peptide amides, substituted peptides, esters and tetrapeptides tested as substrates. Trilysine cleavage is optimal at about pH 8.5, as determined in Tris, borate, or phosphate buffers. Tripeptidase TP activity tested under a number of conditions was not inhibited by soybean trypsin inhibitor (3 mg/ml), phenylmethanesulfonyl fluoride (25 .mu.M), or iodoacetate (9 mM). p-Mercuribenzoate (10 .mu.M), divalent Cu, Co, Ca (2.5 mM), Zn (25 .mu.M) and Hg (10 .mu.M) are inhibitory. Based on Sephadex G-200 chromatography tripeptidase TP has a particle weight of approximately 80,000 daltons. An apparent Km of 5.3 mM was determined for methionylglycylglycine cleavage.