Ro H S, Hong S P, Seo H J, Yoshimura T, Esaki N, Soda K, Kim H S, Sung M H
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
FEBS Lett. 1996 Dec 2;398(2-3):141-5. doi: 10.1016/s0014-5793(96)01222-7.
The beta-strand III formed by amino acid residues Val30-Val36 is located across the active site of the thermostable D-amino acid aminotransferase (D-AAT) from thermophilic Bacillus sp. YM-1, and the odd-numbered amino acids (Tyr31, Val33, Lys35) in the strand are revealed to be directed toward the active site. Interestingly, Glu32 is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in k(cat) value, retaining less than 1% of the wild-type enzyme activity. The k(cat) values of V33A and K35A were changed slightly, but the Km of K35A for alpha-ketoglutarate was increased to 35.6 mM, compared to the Km value of 2.5 mM for the wild-type enzyme. These results suggested that the positive charge at Lys35 interacted electrostatically with the negative charge at the side chain of alpha-ketoglutarate. Site-directed mutagenesis of the Glu32 residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu32-substituted enzymes, the Glu32 residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5'-phosphate (PLP) and the epsilon-amino group of the Lys145 residue.
由氨基酸残基Val30-Val36形成的β-链III横跨嗜热芽孢杆菌YM-1的热稳定D-氨基酸转氨酶（D-AAT）的活性位点，并且该链中的奇数氨基酸（Tyr31、Val33、Lys35）被发现指向活性位点。有趣的是，Glu32也指向活性位点。我们首先通过丙氨酸扫描诱变研究了这些氨基酸残基在催化中的作用。Y31A和E32A突变酶的k(cat)值显著降低，保留的野生型酶活性不到1%。V33A和K35A的k(cat)值略有变化，但K35A对α-酮戊二酸的Km增加到35.6 mM，而野生型酶的Km值为2.5 mM。这些结果表明，Lys35处的正电荷与α-酮戊二酸侧链上的负电荷发生静电相互作用。对Glu32残基进行定点诱变以详细证明该残基的作用。从Glu32取代酶的动力学和光谱特征来看，Glu32残基似乎与磷酸吡哆醛（PLP）的醛基与Lys145残基的ε-氨基之间形成的席夫碱上的正电荷相互作用。
