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 catalysing the conjugation of glutathione (GSH) to endogenous and exogenous electrophilic molecules, with 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 behaviour of the enzyme at 5 °C can be accounted for by its pre-steady-state behaviour if the presence of a natively activated subpopulation (~ 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, k _cat /K _M , is strongly dependent on substrate reactivity (Hammett value 4.2), underscoring that glutathione transferases function as efficient and responsive interception catalysts. The temperature behaviour of the enzyme was also analysed. Both the K _M and K _D values decreased with increasing temperature, while the chemical step k ₃ displayed modest temperature dependence (Q ₁₀ : 1.1-1.2), mirrored in that of the nonenzymatic reaction (Q ₁₀ : 1.1-1.7). Unusually high Q ₁₀ values for GSH thiolate anion formation (k ₂ : 3.9), k _cat (2.7-5.6) and k _cat /K _M (3.4-5.9) support that large structural transitions govern GSH binding and deprotonation, which limits steady-state catalysis.
(© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)