Significant improvement to the catalytic properties of aspartate aminotransferase: role of hydrophobic and charged residues in the substrate binding pocket.
Significant improvement to the catalytic properties of aspartate aminotransferase: role of hydrophobic and charged residues in the substrate binding pocket.
复制标题
天冬氨酸转氨酶催化特性的显着改善：底物结合袋中疏水性和带电残基的作用。
DOI：
10.1021/bi00167a012
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
K. Kirschner
中科院分区：
文献类型：
--
作者：
E. Koehler;M. Seville;J. Jaeger;I. Fotheringham;Michael Hunter;Mark Edwards;J. Jansonius;K. Kirschner
通过将大肠杆菌酪氨酸转氨酶（eTAT）的Leu 39、Glu 141和Arg 293三个关键性不同的残基转移到天冬氨酸转氨酶（eAAT）的等价位置上，检测了eTAT的底物特异性。这些残留物不直接参与催化过程。单突变体eAAT V39 L不仅对酪氨酸而且对天冬氨酸和谷氨酸具有更大的kcat/KM值。相比之下，双突变体eAAT P141 E、A293 R以及三突变体eAAT V39 L、P141 E、A293 R表现出较小的kcat/KM变化。酪氨酸氨基转移酶的匡威突变体，其中eAAT（Val 39）和线粒体AAT（Ala 39，Val 37）的关键残基被转移到eTAT的等同位置，表现出普遍降低的二羧酸和芳香族底物的kcat/KM值。基于eAAT和eAAT V39 L的已知结构以及eTAT的精细模型，这些结果表明eAAT和eTAT的不同底物特异性是由于多个侧链差异和骨架的微小重排。突变体eAAT V39 L的催化效率普遍提高似乎是由于间接效应，即，在底物结合时促进活性位点的闭合。
The substrate specificity of tyrosine aminotransferase (eTAT) from Escherichia coli has been tested by transferring the critically different residues Leu39, Glu141, and Arg293 into equivalent positions of aspartate aminotransferase (eAAT). These residues are not directly involved in the catalytic process. The single mutant eAAT V39L possesses greater values of kcat/KM not only for tyrosine but also for aspartate and glutamate. In contrast, the double mutant eAAT P141E,A293R and also the triple mutant eAAT V39L,P141E,A293R exhibit smaller changes of kcat/KM. The converse mutants of tyrosine aminotransferase, in which critical residues of eAAT (Val39) and of mitochondrial AAT (Ala39, Val37) were transferred into equivalent positions of eTAT, exhibited generally decreased values of kcat/KM for both dicarboxylic and aromatic substrates. On the basis of the known structures of eAAT and eAAT V39L as well as of a refined model of eTAT, these results indicate that the different substrate specificities of eAAT and eTAT are due to multiple side chain differences and minor rearrangements of the backbone. The generally improved catalytic efficiency of the mutant eAAT V39L appears to be due to an indirect effect, namely, the facilitated closure of the active site upon substrate binding.