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Microsomal glutathione transferase 1 (MGST1) has a unique ability to be activated, ≤30-fold, by modification with sulfhydryl reagents. MGST1 exhibits one-third-of-the-sites reactivity toward glutathione and hence heterogeneous binding to different active sites in the homotrimer. Limited turnover stopped-flow kinetic measurements of the activated enzyme allowed us to more accurately determine the K D for the "third" low-affinity GSH binding site (1.4 ± 0.3 mM). The rate of thiolate formation, k 2 (0.77 ± 0.06 s -1 ), relevant to turnover, could also be determined. By deriving the steady-state rate equation for a random sequential mechanism for MGST1, we can predict K M , k cat , and k cat /K M values from these and previously determined pre-steady-state rate constants (all determined at 5 °C). To assess whether the pre-steady-state behavior can account for the steady-state kinetic behavior, we have determined experimental values for kinetic parameters at 5 °C. For reactive substrates and the activated enzyme, data for the microscopic steps account for the global mechanism of MGST1. For the unactivated enzyme and more reactive electrophilic substrates, pre-steady-state and steady-state data can be reconciled only if a more active subpopulation of MGST1 is assumed. We suggest that unactivated MGST1 can be partially activated in its unmodified form. The existence of an activated subpopulation (approximately 10%) could be demonstrated in limited turnover experiments. We therefore suggest that MSGT1 displays a preexisting dynamic equilibrium between high- and low-activity forms.

Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation