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

Abstract

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, 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 behaviour of the enzyme was also analysed. 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 nonenzymatic 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.