Abstract
The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg(2+) or Mn(2+) capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with α-ketoglutarate (α-Kg) binding prior to acetyl-CoA (AcCoA) with Mn(2+), whereas it is steady state random with Mg(2+), suggesting a difference in the competence of the E·Mn·α-Kg·AcCoA and E·Mg·α-Kg·AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d(3)) in the presence of Mg(2+) is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg(2+) or Mn(2+) as the divalent metal ion, whereas V/K(α-Kg) (with Mn(2+)) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as α-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.
Highlights
MATERIALS AND METHODSChemicals—␣-Ketoglutarate, AcCoA, CoA, 2,6-dichlorophenolindophenol (DCPIP), oxaloacetate, oxalate, EDTA, and Chelex 100 were from Sigma
At high concentrations of CoASH, curved double reciprocal plots were observed with ␣-Kg varied (Fig. 4A)
The CoASH inhibition patterns differ from those found for ScHCS, which exhibits noncompetitive inhibition by
Summary
Chemicals—␣-Ketoglutarate, AcCoA, CoA, 2,6-dichlorophenolindophenol (DCPIP), oxaloacetate, oxalate, EDTA, and Chelex 100 were from Sigma. Solvent deuterium isotope effects were obtained by direct comparison of initial rates as a function of ␣-Kg at different fixed levels of AcCoA in H2O and D2O over the pH(D) range 6 –9 (around the pH independent region of the V (or V/K) pH rate profile). For these studies, all reagents including buffers, substrates, and DCPIP were first dissolved in a small amount of D2O, lyophilized overnight to remove H2O and re-dissolved in the required volume of D2O. In Equation 10, y is the value of 1/Ki,Lys at any pH, YL and YH are the pH independent values of 1/Ki,Lys at low and high pH, respectively, and the value 10Ϫ9.5 is the acid dissociation constant for the ␣-amine of lysine
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