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Multicatalytic, High‐Mr Endopeptidase from Postmortem Human Brain
Author(s) -
McDermott J. R.,
Gibson A. M.,
Oakley A. E.,
Biggins J. A.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02045.x
Subject(s) - endopeptidase , chemistry , hydrolysis , peptide bond , biochemistry , tripeptide , peptide , enzyme kinetics , enzyme , neprilysin , residue (chemistry) , stereochemistry , active site
The main high molecular weight (650K) multica‐talytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24‐3 IK and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl‐Gly‐GIy‐Phe‐7‐amido‐4‐methylcoumarin, benzyloxycarboxyl ‐ Gly ‐ Gly ‐Arg‐7‐amido‐4‐methylcoumarin, and benzyloxycarboxyl‐Leu‐Leu‐Glu‐2‐naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P, position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neu‐ropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance‐P, down to 2 nmol/min/mg for bradykinin. The enzyme also had pro‐teinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr 5 ‐Gly 6 bond in luteinizing hormone‐releasing hormone, the K m was 0.95 m M and the specificity constant ( k cat / K m ) was 4.7 ± 103 M ‐l s ‐1 . The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P 1 , and either a small (e.g., ‐Gly or ‐NH 2 ) or hydrophobic residue at P 1 . Hydrolysis of ‐Glu‐X bonds (most notably in neuropeptide Y) and the Arg 6 ‐Arg 7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.