Cawley N X, Chen H C, Beinfeld M C, Loh Y P
Laboratory of Developmental Neurobiology and the Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Feb 23;271(8):4168-76. doi: 10.1074/jbc.271.8.4168.
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.
在体外测定了YAP3p对多种激素原底物切割单碱性和双碱性残基加工位点的特异性及相对效率，这些底物包括：人促肾上腺皮质激素（ACTH）1 - 39、牛胰岛素原、猪胆囊收缩素33、胆囊收缩素（CCK）13 - 33、强啡肽A（1 - 11）、强啡肽B（1 - 13）和酰胺化脑啡肽。YAP3p从ACTH1 - 39生成了ACTH1 - 15。它在胰岛素原的B - C连接和C - A连接的双碱性残基位点进行切割。分别从强啡肽A和强啡肽B生成了亮氨酸脑啡肽 - 精氨酸和亮氨酸脑啡肽 - 精氨酸 - 精氨酸。YAP3p从酰胺化脑啡肽生成了甲硫氨酸脑啡肽 - 赖氨酸 - 赖氨酸，表明该酶可在单个赖氨酸残基处进行切割。CCK33在赖氨酸23和精氨酸9处被切割，在P6和P5位置分别含有一个上游精氨酸残基。各种底物的米氏常数（Km）值在10^(-4)到10^(-5) M之间，对ACTH1 - 39的四碱性位点表现出最高亲和力（1.8×10^(-5) M）。ACTH1 - 39的四碱性残基位点切割效率最高（催化常数与米氏常数的比值kcat/Km = 3.1×10^6 m^(-1) s^(-1)），而CCK13 - 33的单碱性位点和胰岛素原B - C连接的双碱性位点切割效率较低，分别为4.2×10^4 m^(-1) s^(-1)和1.6×10^4 m^(-1) s^(-1)。
