title: The core of allosteric motion in Thermus caldophilus L-lactate dehydrogenase.
abstract: For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S(0.5) value 10(3)-fold and increased the V(max) value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus L-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V(max) 4-fold but reduced pyruvate S(0.5) only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V(max), but 10(2)-reduced pyruvate S(0.5), and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Ikehara
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Arai
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Nayuta
authors.initials: N
authors.lastname: Furukawa
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ohno
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Miyake
authors.affiliation: the Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, and.
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: the Department of Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan.
authors.forename: Akimasa
authors.initials: A
authors.lastname: Miyanaga
authors.affiliation: From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, htaguchi@rs.noda.tus.ac.jp.
authors.forename: Hayao
authors.initials: H
authors.lastname: Taguchi
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D005635:Fructosediphosphates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D019344:Lactic Acid / Q000737:chemistry; D008969:Molecular Sequence Data; D009038:Motion; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019289:Pyruvic Acid / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013824:Thermus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D005635:Fructosediphosphates; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate
keywords: Allosteric Regulation; Dehydrogenase; Enzyme Mutation; Enzyme Structure; Lactic Acid; Thermophile