Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side.
Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side.
复制标题
猪胞质天冬氨酸转氨酶氨基末端区域的结构和功能作用。
DOI：
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发表时间：
1991
影响因子：
4.8
通讯作者：
Yoshimasa Morino
中科院分区：
文献类型：
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作者：
Y. Fukumoto;S. Tanase;F. Nagashima;Shoichi Ueda;K. Ikegami;Yoshimasa Morino
在猪细胞溶质天冬氨酸氨基转移酶（一种二聚体酶）中，锚定在相邻亚基上的氨基末端区域与相邻的松软肽段（残基12-47）连接，松软肽段是小结构域的组成部分，其在底物结合时的轻易移动是该酶的显著“诱导配合”特征。为了评估氨基末端区域对小结构域运动和蛋白质稳定性的贡献，通过使用阿托伐他汀定向体外诱变制备了一系列在氨基末端侧（残基1-9）截短的酶衍生物。残基1-3的缺失对催化活性和热稳定性没有影响。在位置5处具有额外甲硫氨酸的Del 1-5突变酶仅显示野生型酶的kappa cat值（在总转氨作用中）的43%。进一步缺失至残基9导致kappa cat值略微降低。Del 1-9突变体酶仍然保留了野生型酶的33%的kappa cat值。缺失残基1-9后，天冬氨酸和2-氧戊二酸的Km值急剧增加。因此，Del 1-9突变酶显示出kappa cat/Km值的显著降低，仅为野生型酶的2%。氨基末端残基1-9的缺失也导致热稳定性的大幅降低和对蛋白酶401的有限蛋白水解的敏感性增强，已知蛋白酶401在野生型酶的Leu 20处裂解。这些发现表明，增加的构象自由度的软段（残基12-47）的锚定区的大部分损失后，将发生，从而呈现出熵的构象变化，促进底物结合的高亲和力的障碍。
In porcine cytosolic aspartate aminotransferase, a dimeric enzyme, the amino-terminal region anchoring onto the neighboring subunit is linked to the adjoining floppy peptide segment (residues 12-47), an integral part of the small domain whose facile movement upon substrate binding is a striking "induced fit" feature of this enzyme. To assess the contribution by the amino-terminal region to small domain movement and protein stability, a series of enzyme derivatives truncated on the amino-terminal side (residues 1-9) was prepared by using oligonucleotide-directed in vitro mutagenesis. Deletion of residues 1-3 showed no effect on catalytic activity and heat stability. Del 1-5 mutant enzyme with an extra methionine at position 5 showed only 43% of the kappa cat value (in the overall transamination) of the wild-type enzyme. Further deletion up to residue 9 resulted in a slight decrease in kappa cat values. Del 1-9 mutant enzyme still retained a kappa cat value of 33% that of wild-type enzyme. Km values for aspartate and 2-oxoglutarate increased sharply upon deletion of residues 1-9. Accordingly, Del 1-9 mutant enzyme showed a striking decrease in the kappa cat/Km value, to only 2% of that for the wild-type enzyme. Deletion of amino-terminal residues 1-9 resulted also in a large decrease in thermostability and in an enhanced susceptibility to limited proteolysis by protease 401, which is known to cleave at Leu20 of the wild-type enzyme. These findings indicate that an increase in the conformational freedom of the floppy segment (residues 12-47) would occur upon the loss of most of the anchorage region, thereby presenting an entropic barrier to conformational changes that facilitate substrate binding with high affinity.