Structure and Function of Lysine Dehydrogenase

赖氨酸脱氢酶是由底物L-赖氨酸激活的独特酶。L-赖氨酸诱导二聚体酶结合成四聚体。该酶除了催化位点外，还具有效应物结合位点。为了了解催化位点和效应子结合位点中的氨基酸残基，使用各种试剂对二聚体和四聚体酶进行化学修饰。结果表明，该酶的催化位点存在精氨酸和色氨酸残基，效应子结合位点存在赖氨酸和半胱氨酸残基，根癌农杆菌中存在Ti质粒，但该酶的结构基因不是由Ti质粒编码的。用限制性内切酶HindIII部分消化该细菌的染色体DNA，并将其克隆到E. coliJM 109中构建了重组质粒pUC 18。从4，000个克隆细胞中选择一个显示酶活性的克隆细胞。从该细胞中获得的质粒pKUKD 19具有插入的DNA片段（约3.5kb）。通过亚克隆得到含有SspI-XhoI片段（约1.3kb）的pKUKD 19 -4。将该片段插入到高效表达载体pKK 223 -3中，并在E.获得了高产该酶的大肠杆菌细胞。对DNA片段进行测序并确定酶的一级结构。NAD结合结构域位于N端区域，催化结构域位于C端区域。在该酶中没有发现在其它氨基酸脱氢酶中起催化作用的赖氨酸残基（G-G-G-K）。

Lysine dehydrogenase is a unique enzyme which is activated by the substrate L-lysine. L-Lysine induced association of the dimeric enzymes into tetramers. The enzyme has effector-binding sites in addition to the catalytic site. In order to know amino acid residues in the catalytic site and the effector-binding site, chemical modification of the dimeric and tetrameric enzymes were done using various reagents. The results suggest that arginine and tryptophan residues are present in the catalytic site and lysine and cysteine residues are present in the effector-binding site.Agrobacterium tumefaciens has Ti-plasmids, but the structural gene of this enzyme was not coded by Ti-plasmid. The chromosomal DNA of the bacterium was digested partially with a restriction endonuclease, HindIII and cloned into E. coli JM109 using pUC18 as a vector. One cloned cell which showed the enzyme activity was selected from 4,000 cloned cells. The plasmid obtained from this cell, pKUKD19, has a inserted DNA fragment (about 3.5kb). The pKUKD19-4 which has a SspI-XhoI fragment (about 1.3kb) was derived by subcloning. This fragment was inserted into a high expression vector, pKK223-3 and the E. coli cell which produces the enzyme aboundantly was obtained. The DNA fragment was sequenced and the primary structure of the enzyme was determined. The NAD binding domain was located in the N-terminal region and the catalytic domain was in the C-terminal region. The lysine residue (G-G-G-K) which functions in catalysis in other amino acid dehydrogenases was not found in this enzyme.