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

Research Project

Project/Area Number	60560119
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	発酵・醸造
Research Institution	Kochi University
Principal Investigator	MISONO Haruo Kochi University, Faculty of Agriculture, 農学部, 助教授 (30027073)
Co-Investigator(Kenkyū-buntansha)	NAGASAKI Susumu Kochi University, Faculty of Agriculture, 農学部, 教授 (90036690)
Project Period (FY)	1985 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥1,800,000 (Direct Cost: ¥1,800,000) Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000) Fiscal Year 1985: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywords	Lysine Dehydrogenase / Agrobacterium tumefaciens / Activation Of The Enzyme / Dissociation And Association Of The Enzyme / Stereochemistry Of The Enzyme Reaction / 酵素反応の立体化学 / L-リジンの酵素的定量法
Research Abstract	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.