Catalytic Function and Application of A Novel L-Lysine Dehydrogenase from Bacteria.

L-赖氨酸脱氢酶是一种新型的氨基酸脱氢酶，催化L-赖氨酸末端氨基的氧化脱除。我们从根癌农杆菌ICR 1600中纯化了该酶，并对其酶学和理化性质进行了表征，重点研究了该酶与亚基相互作用的催化功能。该酶由两个分子量相同的亚基组成（约39，000）。该酶在赖氨酸存在下形成四聚体，并表现出比二聚体更高的活性。通过用DTNB处理酶来防止酶的缔合和活化。DTNB处理过的酶与2-巯基乙醇的孵育恢复了酶的缔合能力和被L-赖氨酸激活的能力。我们筛选了酶的激活剂，以了解该酶中存在的调节位点。除了L-赖氨酸，许多氨基酸和有机酸，这不是底物和抑制剂，激活酶。这些结果表明，在酶的调节位点。氨基酸和具有大于6个碳长度和羧基的有机酸是有效的活化剂。因此，在酶的调控位点上必须存在一个带正电荷的疏水区。测定了L-赖氨酸脱氢酶催化L-赖氨酸脱氢反应的立体化学。该酶立体特异性地去除L-赖氨酸的pro-R氢。烟酰胺核苷酸依赖性脱氢酶对从还原型辅酶的烟酰胺部分的C-4除去氢显示A或B立体特异性。L-赖氨酸脱氢酶反应中，底物的氢被转移到NADH的二氢烟酰胺环C-4的pro-R位上，该酶具有A-立体专一性。

L-Lysine dehydrogenase is a novel amino acid dehydrogenase which catalyzes the oxidative removal of the terminal amino group of L-lysine. We purified the enzyme from Agrobacterium tumefaciens ICR 1600 and characterized the enzymological and physicochemical properties of the enzyme with emphasis on the catalytic function of the enzyme in relation to the subunit interaction. The enzyme consists of two subunits identical in molecular weight (approximately 39,000). The enzyme associated into tetramer in the presence of L-lysine and showed the higher activity than that of dimer. The association and activation of the enzyme was prevented by treatment of the enzyme with DTNB. The incubation of the DTNB-treated enzyme with 2-mercaptoethanol recovered the ability of association and activation of the enzyme by L-lysine. We screened activators of the enzyme to know the presence of the reguratory site in this enzyme. In addition to L-lysine, many amino acids and organic acids, which were not substrates and inhibitors, activated the enzyme. These results suggest that a regulatory site is present in the enzyme. Amino acids and organic acids having both more than 6 carbon length and carboxyl group were potent activators. Thus, a positive charge and hydrophobic region must be present in the regulatory site of the enzyme.The stereochemistry of the dehydrogenation of L-lysine catalyzed by L-lysine dehydrogenase was determined. The enzyme removed stereospecifically the pro-R hydrogen of L-lysine. Nicotinamide nucleotide-dependent dehydrogenases show A or B stereospecificity for hydrogen removal from C-4 of the nicotinamide moiety of the reduced coenzyme. In the L-lysine dehydrogenase reaction, the hydrogen of the substrate is transferred to the pro-R position at C-4 of the dihydronicotinamide ring of NADH; the enzyme is A-stereospecific.We developed the spectrophotometric procedure for specific microdetermination of L-lysine with the enzyme.