Increased hydrophobicity of the S′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis
Increased hydrophobicity of the S′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis
复制标题
通过定点诱变获得的羧肽酶 Y 中 S1 结合位点的疏水性增加
DOI：
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发表时间：
1985
期刊：
影响因子：
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通讯作者：
K. Breddam
中科院分区：
文献类型：
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作者：
J. Winther;M. Kielland;K. Breddam
酵母羧肽酶Y的Met-398的化学修饰已经被证明影响了该酶相对于P‘1位的专一性。为了确认和进一步研究Met-398的作用，通过定点突变的方法，将该残基替换为亮氨基残基。以含有所需突变的十二核苷酸为引物，在噬菌体M13中对羧基肽酶Y结构基因Prc1的一个亚克隆片段进行诱变，用于体外二次链合成。发现了一个羧基肽酶Y基因398位密码子由ATG突变为TTG的克隆，而亚克隆区域的其余部分是保守的。用亲和层析法分离得到突变酶‡-398CPD-Y，其分子量、N-末端氨基酸序列和含糖量与羧肽酶Y相同。溴化氰降解证实没有Met-398的存在。与野生型酶相比，Leu-398-羧基肽酶Y对底物的亲和力增强，在P‘1位具有较大的疏水基团。这与亮氨酰侧链比甲硫基侧链略小且疏水性更强是一致的。
Chemical modification of Met-398 in carboxypeptidase Y from yeast has previously been shown to affect the specificity of the enzyme with respect to the P′1 position. To confirm and further study the role of Met-398, this residue was substituted by a leucyl residue by means of site-directed mutagenesis. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment ofPRC1, the structural gene for carboxypeptidase Y, using a dodecanucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of the carboxypeptidase Y gene had been mutated from ATG to TTG while the rest of the subcloned region was conserved. This sequence was then reintroduced into the originalPRC1 gene context and a ‡prc1 yeast strain was transformed with the resulting plasmid DNA.The mutant enzyme, Leu-398-carboxypeptidase Y, was isolated by affinity chromatography and shown to have the same molecular weight, N-terminal amino acid sequence and sugar content as carboxypeptidase Y. Cyanogen bromide degradation confirmed the absence of Met-398.Leu-398-CPD-Y was characterized kinetically using a series of N-blocked dipeptide and ester substrates with varying groups in the P′1 position. Compared with the wild type enzyme Leu-398-carboxypeptidase Y showed an increased affinity towards substrates with bulky and hydrophobic groups in the P′1 position. This is consistent with the leucyl side-chain being slightly smaller and more hydrophobic than the methionyl side-chain.