The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178.
The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178.
复制标题
通过亮氨酸 178 的突变替换，羧肽酶 Y 对具有碱性 P1 氨基酸残基的底物的活性显着增加。
作者：
K. Olesen;U. Mortensen;S. Aasmul;M. Kielland;S. Remington;K. Breddam
先前对羧肽酶Y的随机诱变研究表明Leu178位于S1结合袋中，并且后来通过三维结构证实了这一点。我们在这里报告了 Leu178 被 Trp、Phe、Ala、Ser、Cys、Asn、Asp 或 Lys 的突变替换，以及每个突变体的动力学特征，使用在 P1 位置系统变化的底物。这些取代的一般效果是，对于 P1 位上具有不带电荷的氨基酸残基的底物，kcat/Km 降低，对于具有酸性残基的底物几乎没有影响，而对于具有碱性氨基酸残基的底物，则增加 kcat/Km。具有不带电 P1 侧链的底物的 kcat/Km 降低与 178 位残基的性质之间存在明显的相关性。当 Leu178 被另一个疏水性氨基酸残基取代时，观察到较小的降低，当它被极性残基取代时，观察到较大的降低，当它被带电残基取代时，观察到非常大的降低。当 Leu178 被 Asp 取代时，对于 P1 位上 Val 的底物，kcat/Km 降低了 2200 倍。当 Leu178 被带相反电荷的氨基酸残基取代时，带电 P1 侧链底物水解的 kcat/Km 值增加，而当被带相同电荷的残基取代时，kcat/Km 值降低。令人惊讶的是，所有突变体（L178K 除外）都表现出与具有基本 P1 侧链的底物增加的活性。（摘要截断为 250 字）
A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure. We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position. The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues. There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178. A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue. When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position. The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
影响因子：
56.9
作者：
HEDSTROM, L;SZILAGYI, L;RUTTER, WJ
通讯作者：
RUTTER, WJ
酵母羧肽酶 Y 需要糖基化才能实现有效的细胞内转运，但不需要糖基化来实现液泡分选、体内稳定性或活性。
DOI：
10.1111/j.1432-1033.1991.tb15959.x
发表时间：
1991
期刊：
European journal of biochemistry
影响因子：
--
作者：
Winther,JR;Stevens,TH;Kielland-Brandt,MC
通讯作者：
Kielland-Brandt,MC