A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides.
A metalloendopeptidase, optimally active at a neutral pH, was purified from the soluble fraction of brain homogenates. The enzyme (molecular weight about 67000) is strongly inhibited by metal chelators such as EDTA and o-phenanthroline. An EDTA-treated enzyme can be reactivated by several divalent metal ions including Zn2+, Co2+ and Mn2+. The specificity and kinetic parameters of the enzyme were studied with a series of model synthetic substrates. The enzyme preferentially cleaves peptide bonds in which the carbonyl group is contributed by an aromatic amino acid residue in the P1 position. The lowest Km values and the highest Kcat/Km ratios were obtained with substrates having aromatic residues in the P'3 and P1 position or in the P'3 and both the P1 and P2 positions. Lower kcat/Km ratios were obtained with substrates having arginine residues in position P1, and even lower values with those substrates having a glycine or aspartyl residue in this position. Introduction of a D-amino acid residue in either position P1 or P'1 renders the substrate totally resistant to hydrolysis. The specificity studies suggest that the active site of the metalloendopeptidase can accommodate at least five amino acid residues, with two of those residues binding on the N-terminal side and three binding on the C-terminal side of the hydrolyzed bond. Several biologically active peptides are cleaved by the enzyme at sites consistent with the specificity deduced from studies with model synthetic substrates.
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008666:Metalloendopeptidases; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002413:Cations, Divalent; D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases; D008666:Metalloendopeptidases