Publication Abstract from PubMed
For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax value 4-fold at 30 degrees C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than the Thermus thermophilus enzyme (TtLDH), 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 A resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172 to 185) and MR2 (212 to 220), form a compact core for allosteric motion, and His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K and A235R replacements increased Vmax 4-fold, but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, 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 A, 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.
The core of allosteric motion in Thermus caldophilus L-lactate dehydrogenase.,Ikehara Y, Arai K, Furukawa N, Ohno T, Miyake T, Fushinobu S, Nakajima M, Miyanaga A, Taguchi H J Biol Chem. 2014 Sep 25. pii: jbc.M114.599092. PMID:25258319[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
