Role of the prodomain in folding and secretion of rat pancreatic carboxypeptidase A1.

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.

胰羧肽酶A1（CPA1）作为一种无活性的前体——原CPA1被合成，它通过蛋白水解去除95个氨基酸的N端前结构域而加工成为有活性的酶。纯化的大鼠原CPA1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。当原CPA1与α因子信号序列融合替代其内源信号序列时可在酵母中分泌，而成熟的CPA1则不分泌，并且在细胞内被截留和降解。因此，除了使CPA1保持无活性状态外，前结构域还促进了酶原的折叠和分泌。通过反式向CPA1提供前结构域并不能恢复这些功能。猪原CPA的三维结构显示前结构域和酶活性位点之间存在大量广泛的接触，这解释了它的抑制特性[瓜施等人（1992年）《分子生物学杂志》224卷，141 - 157页]。在这些接触中，有在Arg - 71和Asp - A36以及Arg - 124和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原CPA1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原CPA1的成功折叠可能也很重要。