Phillips M A, Rutter W J
Department of Pharmacology, University of Texas Southwestern Medical Center 75235-9041, USA.
Biochemistry. 1996 May 28;35(21):6771-6. doi: 10.1021/bi960113o.
Pancreatic carboxypeptidase A1 (CPA1) is synthesized as an inactive precursor, proCPA1, which is processed to the active enzyme by the proteolytic removal of the 95-amino acid N-terminal prodomain. Purified rat proCPA1 is renatured in vitro after denaturation in guanidine or in guanidine plus reducing agents. In contrast, purified CPA1 is not renatured under any of the conditions tested. While proCPA1 is secreted in yeast when fused to the alpha-factor signal sequence in place of its endogenous signal sequence, mature CPA1 is not secreted and is trapped and degraded intracellularly. Thus, in addition to maintaining CPA1 in the inactive state, the prodomain promotes folding and secretion of the proenzyme. Neither of these functions can be restored by supplying the prodomain to CPA1 in trans. The three-dimensional structure of porcine proCPA reveals a number of extensive contacts made between the prodomain and the enzyme active site which account for its inhibitory properties [Guasch et al. (1992) J. Mol. Biol. 224, 141-157]. Among these contacts are salt bridges formed between Arg-71 and Asp-A36 and between Arg-124 and Asp-A89. Mutation of any of these four residues inhibits secretion of proCPA1 from yeast and results in its intracellular degradation. The effect of the mutations on secretion suggests that interactions which stabilize the binding of prodomain to the native enzyme active site may also be important for the successful folding of proCPA1.
胰腺羧肽酶A1（CPA1）最初以无活性的前体形式合成，即proCPA1，通过蛋白水解去除95个氨基酸的N端前结构域后加工成活性酶。纯化的大鼠proCPA1在胍或胍加还原剂中变性后可在体外复性。相比之下，纯化的CPA1在任何测试条件下都不能复性。当proCPA1与α-因子信号序列融合以取代其内源性信号序列时，它可在酵母中分泌，而成熟的CPA1则不分泌，被困在细胞内并被降解。因此，除了将CPA1维持在无活性状态外，前结构域还促进了酶原的折叠和分泌。通过反式提供前结构域给CPA1，这两种功能都无法恢复。猪proCPA的三维结构揭示了前结构域与酶活性位点之间的大量广泛接触，这解释了其抑制特性[瓜施等人（1992年）《分子生物学杂志》224卷，141 - 157页]。这些接触包括Arg - 71与Asp - A36以及Arg - 124与Asp - A89之间形成的盐桥。这四个残基中的任何一个发生突变都会抑制proCPA1从酵母中的分泌，并导致其在细胞内降解。突变对分泌的影响表明，稳定前结构域与天然酶活性位点结合的相互作用对于proCPA1的成功折叠可能也很重要。
