caa a22 4500 397556934 CHVBK 20180308164805.0 cr unu---uuuuu 161202e199608 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021407 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021407 Involvement of Two Basic Residues (Lys-17 and Arg-39) of Mouse Lung Carbonyl Reductase in NADP(H)-Binding and Fatty Acid Activation: Site-Directed Mutagenesis and Kinetic Analyses [Elektronische Daten] [Masayuki Nakanishi, Mikio Kakumoto, Kazuya Matsuura, Yoshihiro Deyashiki, Nobutada Tanaka, Takamasa Nonaka, Yukio Mitsui, Akira Hara] Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, shows a strong coenzyme preference for NADP(H) over NAD(H), and is uniquely activated by fatty acids. Previous chemical modification and X-ray crystallography studies show that interactions responsible for the coenzyme specificity include salt linkages between the 2′ -phosphate of NADPH and side-chains of Lys-17 and Arg-39 of the enzyme. Although Arg-39 is highly conserved in NADP(H)-dependent enzymes of the SDR family, Lys-17 is substituted with Arg in about half of the NADP(H)-dependent enzymes. The present study shows that mutations of Lys-17 to His (K17H) or Ser (K17S) and of Arg-39 to Ala (R39A) bring about decreases (from 5 to 90-fold) of the affinities for NADP(H), but minor changes in the affinity for NAD+. The binding energy arising from the mutations on the binding of the 2′-phosphate of NADP+ was decreased by 38-66% from the value of 4.8 kcal/mol calculated for the wild-type enzyme. In contrast, the mutation of Lys-17 to Arg (K17R) had little effect on the kinetic or thermodynamic properties. The activation by fatty acids was completely attenuated by the mutations of K17H and K17S, but not by K17R or R39A. These results indicate that the 2′ -phosphate group of NADP(H) is recognized by both Lys-17 and Arg-39, of which Lys-17 is a component of the binding site for the activator, probably interacting with the negatively charged carboxylate group of fatty acids, and also suggest that the existence of a positively charged residue (either Lys or Arg) at position 17 is required for both NADP(H) specificity of the SDR family enzymes and fatty acid activation of the pulmonary carbonyl reductase. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence fatty acid activation nationallicence carbonyl reductase nationallicence NADP(H) specificity nationallicence short-chain dehydrogenase/reductase family nationallicence site-directed mutagenesis nationallicence Nakanishi Masayuki Department of Applied Chemistry, Faculty of Engineering, Gifu University Yanagido, Gifu, Gifu 501-11 aut Kakumoto Mikio Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut Matsuura Kazuya Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut Deyashiki Yoshihiro Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut Tanaka Nobutada Department of BioEngineering, Nagaoka University of Technology Kamitomioka, Nagaoka, Niigata 940-21 aut Nonaka Takamasa Department of BioEngineering, Nagaoka University of Technology Kamitomioka, Nagaoka, Niigata 940-21 aut Mitsui Yukio Department of BioEngineering, Nagaoka University of Technology Kamitomioka, Nagaoka, Niigata 940-21 aut Hara Akira Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut The Journal of Biochemistry Oxford University Press 120/2(1996-08), 257-263 0021-924X 120:2<257 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021407 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021407 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nakanishi Masayuki Department of Applied Chemistry, Faculty of Engineering, Gifu University Yanagido, Gifu, Gifu 501-11 aut NATIONALLICENCE 700 1- Kakumoto Mikio Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut NATIONALLICENCE 700 1- Matsuura Kazuya Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut NATIONALLICENCE 700 1- Deyashiki Yoshihiro Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut NATIONALLICENCE 700 1- Tanaka Nobutada Department of BioEngineering, Nagaoka University of Technology Kamitomioka, Nagaoka, Niigata 940-21 aut NATIONALLICENCE 700 1- Nonaka Takamasa Department of BioEngineering, Nagaoka University of Technology Kamitomioka, Nagaoka, Niigata 940-21 aut NATIONALLICENCE 700 1- Mitsui Yukio Department of BioEngineering, Nagaoka University of Technology Kamitomioka, Nagaoka, Niigata 940-21 aut NATIONALLICENCE 700 1- Hara Akira Biochemistry Laboratory, Gifu Pharmaceutical University 5-6-1 Mitahorahigashi, Gifu, Gifu 502 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/2(1996-08), 257-263 0021-924X 120:2<257 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford