Characterization of the bacterial metalloendopeptidase pitrilysin by use of a continuous fluorescence assay

Abstract

Pitrilysin (EC 3.4.99.44) has been purified from an over-expressing strain of Escherichia coli. A 13-residue quenched-fluorescent-peptide substrate for the enzyme has been synthesized, and found also to be cleaved by the homologous enzyme, insulinase (EC 3.4.99.45). The action of pitrilysin on peptides and proteins was studied: insulin B chain was the most rapidly degraded, small peptides down to 10 residues in length were cleaved more slowly, intact insulin was cleaved very slowly but with a very low Km, and there was no action on the larger proteins tested. Since the activity of pitrilysin is confined to substrates smaller than proteins, it can be described as an endopeptidase of the 'oligopeptidase' type, and like other such enzymes, it did not interact with alpha 2-macroglobulin. The metal-dependence of pitrilysin was confirmed, and it was found to be inhibited by bacitracin, especially in the presence of zinc.

The Preparation of Fluorescence-Quenched Probes for Use in the Characterization of Human Factor Xa Substrate Binding Domains

“…Following the procedure of Anastasi et al [9], diisopropylethylamine (1.5 mL, 8.6 mmol) was added to commercial N 6 -dinitrophenyl-L-lysine (1.50 g, 4.3 mmol) in 1:1 acetonitrile/water (22 mL).…”

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo

“…This motif is an inversion of the more common zinc-binding motif (HEXXH) of other metalloproteases (11). Biochemical properties such as substrate specificity, enzyme kinetics, and metal stoichiometry have been studied (12); however, no obvious phenotypic deficiencies were observed in pitrilysin-deficient E. coli mutants (13), and not much is known about the physiological functions of this enzyme in bacteria. IDE homologs are highly conserved between bacteria and mammals and, in bacteria, they are localized to the cytosol, the plasma membrane, or the periplasm (5).…”

Cleavage of Various Peptides with Pitrilysin fromEscherichia coli: Kinetic Analyses Using β-Endorphin and Its Derivatives

“…1,2) However, the cleavagesites in these peptides remain to be determined, although the oxidized insulin B chain and vasoactive intestinal peptide (VIP) have been reported to be cleaved by the enzyme, predominantly at Tyr 16 -Leu 17 and Leu 13 -Arg 14 , respectively. 1,2) In addition, degradation of the peptides by pitrilysin has not so far been analyzed kinetically, although the binding affinities of insulin, oxidized insulin B chain, and glucagon have been estimated from their K i values for degradation of a quenched-fluorescent substrate by the enzyme. 1) In this study, pitrilysin was overproduced, purified, and analyzed for proteolytic activity using various peptide substrates.…”

“…1,2) It is a periplasmic enzyme 3) and acts in monomeric form. 2,4) The ptr gene encoding this enzyme is located in the chromosome between the recB and recC genes, which encode subunits of endonuclease V. 5) The physiological role of pitrilysin is not known, because gene disruption studies have indicated that this enzyme is not essential for normal cell growth.…”