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.
Highlights
Allostery is one of the important but complicated properties of proteins
The two structures obviously differ from each other in their quaternary structures, the P-axis-related dimers taking on open and closed conformations (Fig. 3A), as in the case of the T and R states of Bifidobacterium longum LDH (BlLDH) (9) and Lactobacillus casei LDH (LcLDH) (16), respectively. This indicates that Thermus caldophilus L-lactate dehydrogenase (TcLDH) has two major conformational states, the T and R states, in the allosteric transition, the enzyme undergoes a more markedly smaller quaternary structural change than BlLDH (3.69 Å) and LcLDH (4.89 Å) do
These two clusters are unique in TcLDH, because the basic residues in them are poorly conserved in other Lactate dehydrogenase (LDH) including LcLDH and BlLDH (Fig. 2)
Summary
Allostery is one of the important but complicated properties of proteins. Results: Structural and kinetic analyses indicated the simple allosteric machinery of Thermus caldophilus L-lactate dehydrogenase (TcLDH). Structural and kinetic analyses revealed the simple and unique allosteric machinery of TcLDH, where the MR1MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule. Thermus caldophilus LDH (TcLDH) is a heat-stable allosteric LDH (21) and shows less than 40% amino acid identity to LcLDH or BlLDH (Fig. 2) This enzyme is uniquely activated through chemical modifications with 2,3-butanedione (22–25), diethyl pyrocarbonate, or acetic anhydride (18) with protection of the catalytic site by NADH and oxamate. The results revealed the simple and unique allosteric machinery of TcLDH
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