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 ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic 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.