Enzymology of reactive intermediate protection: kinetic analysis and temperature dependence of the mesophilic membrane protein catalyst MGST1.

Glutathione transferases (GSTs) are a class of phase II detoxifying enzymes catalyzing the conjugation of glutathione (GSH) to endogenous and exogenous electrophilic molecules, microsomal glutathione transferase 1 (MGST1) being one of its key members. MGST1 forms a homotrimer displaying third-of-the-sites-reactivity and up to 30-fold activation through modification of its Cys-49 residue. It has been shown that the steady-state behavior of the enzyme at 5 °C can be accounted for by its pre-steady-state behavior if the presence of a natively activated subpopulation (approximately 10 %) is assumed. Low temperature was used as the ligand free enzyme is unstable at higher temperatures. Here, we overcame enzyme lability through stop-flow limited turnover analysis, whereby kinetic parameters at 30 °C were obtained. The acquired data are more physiologically relevant and enable confirmation of the previously established enzyme mechanism (at 5 °C), yielding parameters relevant for in vivo modelling. Interestingly, the kinetic parameter defining toxicant metabolism, kcat /KM , is strongly dependent on substrate reactivity (Hammett value 4.2), underscoring that glutathione transferases function as efficient and responsive interception catalysts. The temperature behavior of the enzyme was also analyzed. Both the KM and KD values decreased with increasing temperature, while the chemical step k3 displayed modest temperature dependence (Q10 : 1.1-1.2), mirrored in that of the non-enzymatic reaction (Q10 : 1.1-1.7). Unusually high Q10 values for GSH thiolate anion formation (k2 : 3.9), kcat (2.7-5.6) and kcat /KM (3.4-5.9) support that large structural transitions govern GSH binding and deprotonation, which limits steady state catalysis.