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

Abstract

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, k₂ (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.

Figure 1

Structures of the electrophilic substrates used in this study and their specific reactivity described by Hammett substituent constants for the para position (σ⁻): 2,5-dichloronitrobenzene (2,5-DCNB, σ⁻ = 0.23), 4-chloro-3-nitro-benzamide (CNBAM, σ⁻ = 0.63), 4-chloro-3-nitroacetophenone (CNAP, σ⁻ = 0.87), 1-chloro-2,4-dinitrobenzene (CDNB, σ⁻ = 1.3).

Figure 2

GSH-binding and thiolate formation to activated MGST1 after limited turnover. Limited turnover experiment where GSH-bound MGST1 is rapidly mixed with CDNB at a ratio of approximately 1.5/trimer. Thiolate anion re-formation was recorded after the initial burst (mixing the enzyme with 8 µM CDNB) as an increase in absorbance at 239 nm with 0.25–20 mM GSH. The dependence of k_obs for GSH thiolate formation on GSH concentration was fitted to Equation 1 in GraphPad Prism 5. Measurement values are given ± standard error of the mean. Enzyme concentration used was 5.8 µM trimer as measured by an active site titration (not shown).

Figure 3

Ratios of the calculated vs experimental catalytic constants for (●) K_M, (□) k_cat and (▲) k_cat/K_M. (A) Activated MGST1, saturating GSH and varying electrophile concentrations. (B) Unactivated MGST1, saturating GSH and varying electrophile concentrations. (C) Activated MGST1, saturating electrophile and varying GSH concentrations. (D) Unactivated MGST1, saturating electrophile and varying GSH concentrations.

Figure 4

Representative image of the limited turnover process. The second order rate constant k₄ will be used below for fitting the chemical reaction between EGS⁻ and CDNB and was derived as the initial slope of the fit of CDNB bursts in references^, circumventing any uncertainty in the binding affinity of CDNB.

Figure 5

(A) Unactivated MGST1 limited turnover. Simulation based on assumption that the entire enzyme population is in its unactivated form. k₂ is set to that optimized by the KinTec Explorer program in Figure 5B. (B) Unactivated MGST1 limited turnover. Simulation based on assumption that a fraction of the enzyme population is in an activated form (10 %). k₂ for the unactivated and activated enzyme populations are fit by KinTek Explorer to 0.0055 s⁻¹ and 0.77 s⁻¹ respectively.

Figure 6

Activated MGST1 limited turnover. Simulation based on assumption that the entire enzyme population is in its activated form. k₂ for the activated enzyme is fit by KinTek Explorer to 0.41 s⁻¹.

Figure 7

Hammett plots of steady-state kinetic parameters for four electrophilic substrates 2,5-DCNB, CNBAM, CNAP, CDNB (left to right), and GSH at 5°C. (●) NEM activated MGST1 log k_cat/K_M, (○) Unactivated MGST1 log k_cat/K_M, (■) NEM activated MGST1, log k_cat and (□) Unactivated MGST1 log k_cat. All experiments were performed at 5 °C. (A) Saturating GSH concentration (5 mM) and varying concentration of the electrophile. (B) Saturating concentrations of electrophile (1 mM for all except CDNB which was at 0.5 mM) and varying GSH concentration. Slopes are indicated.

Figure 8

MGST1 representation with subunits in different color showing one GSH molecule covered by a schematic lid. The entry path for hydrophobic electrophiles from the membrane phase is shown (E). Membrane width is indicated by blue lines. Generated with Pymol from PDB entry 2H8A.

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5