Preferential action of rat brain cathepsin B as a peptidyl dipeptidase converting pro-opioid oligopeptides.
Purified rat brain cathepsin B (EC 3.4.22.1) converted prodynorphins or proenkephalins to shorter active forms by the preferential removal of C-terminal dipeptides. The substrate affinities for Met-enkephalin-Arg-Phe or -Arg-Gly-Leu were Km 46 and 117 microM, and kcat/Km ratios were 67 and 115 microM-1, min-1, respectively. Met-Enkephalin was inactivated by the same mechanism (Km-450 microM; kcat/Km = 0.12 microM-1 min-1). The comparison of cathepsin B hydrolysis for pro-opioids, a synthetic hexapeptide and its fragments, C-blocked peptides (pro-opioid amides, Met-enkephalin amide, substance P), and bovine myelin basic protein, provided information on the influence of the C-terminal residues on dipeptide release, the rates as correlated to peptide length, and the optimal arrangement of residues favoring scission at the P1-P'1 sites. The brain enzyme was stereospecific and did not act on peptides with C-terminal D-amino acid substituents. Arg hindered and Pro blocked the release of C-terminal dipeptides when in the P'2 positions. The suppression of dipeptide release by agents inhibiting endopeptidase actions such as E-64 and leupeptin, and the endogenous brain factor (cerebrocystatin) point to similar catalytic mechanisms for the exopeptidase action.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002401:Cathepsin B; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004150:Dipeptidases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism*; D020689:Exopeptidases; D007700:Kinetics; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; C033660:preproenkephalin; D002403:Cathepsins; D010450:Endopeptidases; D020689:Exopeptidases; D010447:Peptide Hydrolases; D004150:Dipeptidases; D002401:Cathepsin B