Multicatalytic, high-Mr endopeptidase from postmortem human brain.
The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-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 P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides 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 proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 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 P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.
McDermott
J R
JR
Medical Research Council, Neurochemical Pathology Unit, Newcastle General Hospital, England.
Gibson
A M
AM
Oakley
A E
AE
Biggins
J A
JA
D001921:Brain / Q000201:enzymology*; D002102:Cadaver; D002384:Catalysis; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D006868:Hydrolysis; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism; D014018:Tissue Distribution
D009479:Neuropeptides; D010450:Endopeptidases