Understanding the catalytic mechanism of the triazine hydrolase from Arthrobacter aurescens TC1

The catalytic mechanism of the Zn(II)-dependent triazine hydrolase (TrzN) from Arthrobacter aurescens TC1 was examined by measuring the pH dependence of the Michaelis constants k_cat, K_m, and k_cat/K_m, the solvent isotope effect, and the thermodynamic parameters of the hydrolysis of atrazine. TrzN was maximally active towards atrazine over the pH range 6.5-10.0, and fits of these data yielded a pK'_ES < 4.5, a pK_ES > 11, and a pK_E value of 6.4 ± 0.2. Based on these data, along with those previously reported, the observed pK'_ES and pK_E values are likely due to the active site residues Glu241 and His274, whereas the observed pK_ES value is possibly due to Tyr215. Proton inventory studies indicated that at least three protons are transferred in the rate-limiting step of the reaction at pD 7.5. An Arrhenius plot was constructed from 278 to 308 K by plotting ln(k_cat) vs. 1/T, providing an E_a of 16.7 ± 0.3 kJ·mol^-1 and $\Delta H^{‡}$ and $\Delta S^{‡}$ values of 14 ± 2 kJ·mol^-1 and -170 ± 10 J·mol^-1 at 25 °C, respectively, resulting in a $\Delta G^{‡}$ of 65.5 ± 0.1 kJ·mol^-1. These data coupled with molecular dynamics simulations of wild-type TrzN and the TrzN Glu241Gln mutant provided evidence for the proposed catalytic roles of active site residues, and identified molecular motions associated with substrate binding and allosteric regulators of transition-state arrangement. Taken together, these data support the proposed catalytic mechanism for the hydrolytic dehalogenation of atrazine by TrzN.