Conformational equilibrium of an enzyme catalytic site in the allosteric transition

Abstract

The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase

“…8). This structural observation is well consistent with the results obtained on chemical modification (22)(23)(24)(25) or amino acid replacement of the basic amino acid residues (12,13,25), because such modifications consistently reduce the static repulsion. In addition, the structural comparison explains the distinct allosteric properties of TcLDH and TtLDH (A154G/ H179Y), because His 179 is located just at the center of MR1, whereas Ala 154 is located in the ␣1F-␤F loop on the protein surface.…”

“…Enzyme Assays and Protein Concentration DeterminationIt is known that TcLDH shows sigmoidal saturation curves for substrates in the absence of FBP (12,(21)(22)(23)(24)(25), but no significant cooperativity on NADH binding, giving dissociation constants of 0.9 -1.2 M for NADH independently of FBP or the P mutations (25 …”

“…This L value is markedly greater than the value for LcLDH (3.0 ϫ 10 2 ) (16) and is reduced to 6.8 by the P2 mutation (R173Q/R216L) (25). Recently, the apo and holo structures of Thermus thermophilus LDH (TtLDH), which only has the A154G and H179Y replacements of TcLDH, were determined (27).…”

“…This enzyme is uniquely activated through chemical modifications with 2,3-butanedione (22)(23)(24)(25), diethyl pyrocarbonate, or acetic anhydride (18) with protection of the catalytic site by NADH and oxamate. Arg 173 (12) and Arg 216 (25) are predominantly modified with 2,3-butandione, and the R173Q and R216L replacements (P mutations) also markedly enhance the FBP-independent activity of TcLDH (12,25). These observations strongly suggest that the inactive state (T state) of TcLDH is stabilized by the positively charged groups within the protein.…”

“…The allosteric constant (L ϭ [T state]/[R state]) was estimated to be 1.8 ϫ 10 5 for TcLDH on kinetic analysis of the lactate oxidation reaction at pH 6.75, assuming the MWC-type allosteric transition (25). This L value is markedly greater than the value for LcLDH (3.0 ϫ 10 2 ) (16) and is reduced to 6.8 by the P2 mutation (R173Q/R216L) (25).…”