Structural Feature of the Nucleotide-Binding Site in Enzymes

在此之前，我们已经设计并合成了一种新型的亲和标记试剂，特异性的赖氨酰残基位于蛋白质中的核苷酸或糖核苷酸结合位点，也适用于甘氨酸丰富的区域的氨基酸残基的定点突变。本课题主要研究结果如下：1）通过对马铃薯块茎UDPG焦磷酸化酶的亲和标记，确定了酶活性位点的5个赖氨酰残基（Lys-263、Lys-329、Lys-367、Lys-409和Lys-410）。在这些赖氨酰残基中，Lys-367可能直接参与催化功能，而Lys-329和Lys-263分别在PPi的结合和UDPG与酶结合后的构象变化中发挥作用。2）我们制备了鸡骨骼肌腺苷酸激酶的突变酶，其中Lys-66被各种其他氨基酸取代。基于这些突变酶的性质，我们为Lys-66在识别底物AMP的腺嘌呤环中的作用提供了证据。3）在嗜热脂肪芽孢杆菌亮氨酸脱氢酶的化学修饰中，结果表明，Lys-80参与了该酶的催化功能。4）大肠杆菌F1-ATP酶探测使用反应性ATP类似物，腺苷三磷酸吡哆醛。在不存在Mg^2+的情况下，α-Lys-201和β-Lys-155残基是主要靶点，而在存在Mg ^2+的情况下，在β-Lys-155和β-Lys-211残基中观察到主要标记。结果表明，这三个残基靠近ATP的γ-磷酸基团，与催化位点结合，在Mg^<2+>存在时，两个β残基和γ-磷酸基团变得更接近。

Previously, we have designed and synthesized a new type of affinity labeling reagents specific for the lysyl residue located in the nucleotide- or sugar nucleotide-binding site in proteins, and also applied site-directed mutagenesis for amino acid residues in the glycine-rich region. Under the present title of project, we have obtained the following results.1) By affinity labeling of potato tuber UDPG pyrophosphorylase with reagents having different structures, we have identified five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410) located around the UDPG molecule at the active site of the enzyme. Of these lysyl residues, Lys-367 may participate directly in the catalytic function, whereas Lys-329 and Lys-263 play the roles, respectively, in the binding of PPi and in the conformational changes upon binding of UDPG to the enzyme.2) We have prepared muatant enzymes of chicken skeletal muscle adenylate kinase in which Lys-66 is replaced by various other amino acids. Based on the properties of those mutant enzymes, we provide evidence for the role of Lys-66 in the recognition of the adenine ring of the substrate AMP.3) In the chemical modification of leucine dehydrogenase of Bacillus stearothermophilus, we obtained the results suggesting the participation of Lys-80 in the catalytic function of this enzyme.4) The catalytic site of Escherichia coli F1-ATPase was probed using areactive ATP analogue, adenosine triphosphopyridoxal. In the absence of Mg^<2+>, the alphaLys-201 and betaLys-155 residues were the major target, whereas, in the presence of the ion, predominant labeling was observed in the betaLys-155 and betaLys-211 residues. The results suggest that those three residues are located close together near the gamma-phosphate group of ATP bound to the catalytic site, and that the two beta residues and the gamma-phosphate group become closer to each other in the presence of Mg^<2+>.