Unique cleavage specificity of 'prohormone thiol protease' related to proenkephalin processing.
'Prohormone thiol protease' (PTP) represents the major enkephalin precursor processing activity in chromaffin granules. In this study, cleavage specificity of PTP for paired basic and monobasic residues was examined with a series of model peptide-MCA (-methylcoumarinamide) substrates. Monobasic peptides were cleaved at the COOH- and NH2-terminal sides of the single basic residue. Dibasic peptides, however, were preferentially cleaved at the NH2-terminal side of the pair, or between the two basic residues, with low cleavage at the COOH-terminal side of the pair. Inhibition by the peptide inhibitor (D-Tyr)-Glu-Phe-Lys-Arg-CH2Cl provided further evidence for PTP's specificity for the dibasic Lys-Arg site. Inhibition by Z-Leu-Val-Gly-CHN2 and Z-Arg-Leu-Val-Gly-CHN2 suggests involvement of Val-Gly in substrate binding to PTP; these two cystatin C-related inhibitors also indicate PTP as a cysteine protease. These results demonstrate PTP's unique cleavage specificity that differs from other processing endopeptidases, including the subtilisin-related proprotein convertases, PC1/PC3, and PC2, as well as the pituitary proopiomelanocortin-converting enzyme, PCE. This study provides further evidence for PTP as a novel prohormone processing enzyme that belongs to the class of cysteine proteases.
Azaryan
A V
AV
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.
Hook
V Y
VY
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004745:Enkephalins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D003546:Cysteine Endopeptidases; C083275:prohormone thiol protease