Differential activation of endopeptidase EC 3.4.24.15 toward natural and synthetic substrates by metal ions.
The activity of endopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP 24.15), as measured by cleavage of a quenched fluorescent substrate, 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys (2,4-dinitrophenyl), was increased 2-3 fold by the addition of 1 mM Mn2+ or of 10 mM Ca2+. The inhibitory capability of a specific EP. 24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, was also increased at similar concentrations of these metal ions. However, the hydrolysis of naturally-occurring peptides, thought to be the physiological substrates for EP 24.15, was not affected by either Mn2+ or Ca2+. These results suggest that the binding of synthetic analogs to the enzyme may differ significantly from the binding, and thus hydrolysis, of natural peptide substrates and caution against drawing conclusions about substrate interactions with the active site from data obtained with modified peptide ligands.
Wolfson
A J
AJ
Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
Shrimpton
C N
CN
Lew
R A
RA
Smith
A I
AI
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008345:Manganese; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D015032:Zinc; D002118:Calcium