SPECIFICITY AND KINETIC-STUDIES ON THE CLEAVAGE OF VARIOUS PROHORMONEMONO-PAIRED AND PAIRED-BASIC RESIDUE SITES BY YEAST ASPARTIC PROTEASE-3

The specificity and relative efficiency of cleavage of mono and paired -basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH(1-39), bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A( 1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH(1-15) f rom ACTH(1-39) It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg a nd Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphi n B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorp hin showing that cleavage by this enzyme can occur at a lone pair of L ys residues. CCK33 was cleaved at Lys(23) and Arg(9), each containing an upstream Arg residue at the P6 and P5 position, respectively, K-m v alues were between 10(-4) and 10(-5) M for the various substrates, wit h the highest affinity exhibited for the tetrabasic site of ACTH(1-39) (1.8 x 10(-5) M) The tetrabasic residue site of ACTH(1-39) was cleave d with the highest relative efficiency (k(cat)/K-m = 3.1 x 10(6) M(-1) s(-1)), while that of the monobasic site of CCK13-33 and the paired-b asic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) M(-1) s(-1) and 1.6 x 10(4) M(-1) s(-1), respectively.