Structure and Function of Lysine Dehydrogenase

赖氨酸脱氢酶是一种由底物l -赖氨酸激活的独特酶。赖氨酸诱导二聚体酶结合成四聚体。酶除了催化位点外还有效应结合位点。为了了解催化位点和效应结合位点的氨基酸残基，用不同的试剂对二聚体酶和四聚体酶进行了化学修饰。结果表明，精氨酸和色氨酸残基存在于催化位点，赖氨酸和半胱氨酸残基存在于效应结合位点。农杆菌具有ti质粒，但该酶的结构基因未被ti质粒编码。用限制性内切酶HindIII消化部分细菌染色体DNA，以pUC18为载体克隆到大肠杆菌JM109中。从4000个克隆细胞中选出一个具有酶活性的克隆细胞。从该细胞中获得的质粒pKUKD19插入了一个DNA片段（约3.5kb）。通过亚克隆获得了含有SspI-XhoI片段（约1.3kb）的ppkd19 -4。将该片段插入高表达载体pKK223-3中，获得了大量产生该酶的大肠杆菌细胞。对DNA片段进行测序，确定酶的一级结构。NAD结合域位于n端，催化域位于c端。赖氨酸残基（G-G-G-K）在其他氨基酸脱氢酶中没有催化作用。

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.