The kinetic mechanism of microsomal glutathione transferase 1 (MGST1)

Microsomal glutathione transferase I (MGST1) is a homotrimeric membrane-bound enzyme involved in detoxication of numerous toxic electrophilic compounds, e.g. halogenated alkenes and arenes, alpha, beta-unsaturated hydrocarbons and peroxides. MGST1 is expressed in large amounts in most organs, which speaks for an important role for this enzyme. The study described herein focuses on the kinetic mechanism and activation of MGST1.

One remarkable feature of this enzyme is its ability to be activated upon various chemical treatments. Reaction of a unique cysteine-49 with sulfhydryl reagents increases the rate of catalysis, up to 30-fold, towards reactive second substrates such as 1-chloro-2,4-dinitrobenzene (CDNB). It is possible that activation, which may be caused by substrates, is an alternative rapid response to toxic conditions in the cell. Studies investigating the pKa and reactivity of cysteine-49 showed no altered pKa and reduced reactivity compared to model thiols. However, these experiments gave an indication that this residue is situated in a hydrophobic environment, thus possessing a preference for reaction with hydrophobic electrophiles.

Investigations of the kinetic mechanism have revealed several interesting aspects of MGST1 catalysis. Glutathione (GSH)-thiolate formation is a slow process with this enzyme. The procedure can be described by a two-step mechanism, consisting of rapid formation of an initial pre-complex (E*GSH) followed by slow deprotonation of GSH to the final complex (E * GS-, kGs- ~ 0.4 s-1). When utilizing the N-ethylmaleimide activated enzyme the rate of this step is greatly enhanced (kGs- ~3.6 s-1). Thus the mechanistic basis for activation has been defined. Rapid mixing of MGST1, pre-charged with GSH, with CDNB gave rise to a burst of product formation (kChem ~ 500 s-1). Further analysis of the chemical step, using substrates with varying reactivity, has shown a strong dependence on the relative chemical reactivity. Thus the efficiency of conjugation increases with more reactive substrates, a useful property indeed. Although the efficiency of thiolate formation is changed upon activation of MGST1 the chemical step appears largely unaffected.

Considering the properties of the active site in the trimeric MGST1, the kinetic data taken together with studies on the activation mechanism suggest substoichiometric binding of GSH, i.e. one GSH per trimer of enzyme. This suggests subunit cooperativity. Whether one active site is built up from the three subunits or binding to one active site prevents binding to the other is however not known.

The environmentally sensitive, fluorescent GSH-conjugate of 6-bromoacetyl-2-N,N-dimethylaminonaphthalene has been shown to be an attractive candidate for probing ligand interactions with glutathione transferases (GSTs). In addition, our initial studies revealed that 6-bromoacetyl-2-N,N-dimethylaminonaphthalene was a fluorogenic substrate for GSTs. This led us to synthesize the chloro-analog, which was shown to be a much more sensitive substrate for MGST1, and cytosolic GSTs Alpha, Mu and Pi.

List of scientific papers

I. Svensson R, Rinaldi R, Swedmark S, Morgenstern R (2000). Reactivity of cysteine-49 and its influence on the activation of microsomal glutathione transferase 1: evidence for subunit interaction. Biochemistry. 39(49): 15144-9.
https://pubmed.ncbi.nlm.nih.gov/11106493

II. Morgenstern R, Svensson R, Bernat BA, Armstrong RN (2001). Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1. Biochemistry. 40(11): 3378-84.
https://pubmed.ncbi.nlm.nih.gov/11258959

III. Svensson R, Armstrong RN, Morgenstern R (2003). Kinetic characterization of activated microsomal glutathione transferase 1: Thiolate anion formation, chemical catalysis and solvent kinetic isotope effects. [Manuscript]

IV. Svensson R, Greno C, Johansson AS, Mannervik B, Morgenstern R (2002). Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases. Anal Biochem. 311(2): 171-8.
https://pubmed.ncbi.nlm.nih.gov/12470677