Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1

Biochemistry
Richard SvenssonRalf Morgenstern

Abstract

Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated, up to 30-fold, by the reaction with sulfhydryl reagents, e.g., N-ethylmaleimide. Analysis of glutathione (GSH) thiolate formation, which occurs upon mixing activated MGST1 with GSH, reveals biphasic kinetics, where the rapid phase dominated at higher GSH concentrations. The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (K(D)(GSH) approximately 10 mM), followed by slower formation of thiolate (k(2) approximately 10 s(-1)). The release rate (or protonation of the enzyme GSH thiolate complex) of GS(-) was slow (k(-2) = 0.016 s(-1)), consistent with overall tight binding of GSH. Electrophilic second substrates react rapidly with the E*GS(-) complex, and again, a two-step mechanism is suggested. In comparison to the unactivated enzyme [Morgenstern et al. (2001) Biochemistry 40, 3378-3384], the mechanisms of GSH thiolate formation and electrophile interaction are similar; however, thiolate anion formation is enhanced 30-fold in the activated enzyme, contributing to an increased k(cat) (3.6 s(-1)). Interestingly, in the activated enzyme, thiolate formation and proton release from the enzyme are not strictly...Continue Reading

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Citations

Oct 29, 2010·The Journal of Biological Chemistry·Agnes Rinaldo-MatthisJesper Z Haeggström
Apr 19, 2011·Drug Metabolism Reviews·Ralf MorgensternKatarina Johansson
Apr 8, 2009·Analytical Biochemistry·Johan AlanderRalf Morgenstern
May 7, 2009·Archives of Biochemistry and Biophysics·Johan AlanderRalf Morgenstern
Dec 21, 2014·Biochimica Et Biophysica Acta·Jason M Hansen, Craig Harris
Oct 6, 2012·Biochimica Et Biophysica Acta·Marcel Deponte
Nov 1, 2018·Organic & Biomolecular Chemistry·Zhitao LeiZhigang Xie
May 25, 2006·The Journal of Pharmacology and Experimental Therapeutics·Yanbin Ji, Brian M Bennett
Jan 17, 2020·Critical Reviews in Toxicology·Patrick E Hanna, M W Anders
Nov 30, 2005·The Journal of Biological Chemistry·Yanbin JiBrian M Bennett
Nov 3, 2017·Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan·Aya Shibata
Dec 8, 2004·Archives of Biochemistry and Biophysics·Laura S Busenlehner, Richard N Armstrong

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