A COMPARISON OF THE ENZYMATIC AND PHYSICOCHEMICAL PROPERTIES OF HUMANGLUTATHIONE TRANSFERASE M4-4 AND 3 OTHER HUMAN MU-CLASS ENZYMES

The multigene family of cytosolic glutathione S-transferases (GSTs) co nsists of four classes (Alpha, Mu, Pi, and Theta), all involved in the detoxication of reactive electrophiles. The human Mu class GSTs consi st of at least four expressed isozyme subunits, GST M1, GST M2, GST M3 , and GST M4, which have 70-90% amino acid sequence identity. The gene and cDNA sequences for GST M4 have been determined recently (K. E. Co mstock, K. J. Johnson, D. Rifenbery, and W. D. Henner, J. Biol. Chem. (1993) 268, 16958-16965). Cloning of GST M4 cDNA into an Escherichia c oli expression system permitted the production of the corresponding pr otein. The enzyme was purified and shown to have a relatively low spec ific activity with the standard GST substrate 1-chloro-2,4-dinitrobenz ene (1.4+/-0.2 mu mol min(-1) mg(-1) protein), but an activity equival ent to other Mu class enzymes with other tested substrates. The protei n forms functional dimers composed of subunits with a IM, of approxima tely 26,400. A detailed comparison of the activity with various substr ates and inhibitors was performed between GST M4-4 and other human Mu class GSTs, GST M1a-1a, GST M2-2, and GST M3-3, produced in bacterial expression systems. Despite the high level of amino acid sequence iden tity, the enzymatic properties of these enzymes were quite different. Comparisons with the crystallographic structure of a homologous rat GS T, GST 3-3, indicate that a number of the nonconserved amino acid resi dues can be assigned to the putative active site of GST M4-4. This sug gests that diversification in the evolution of these genes has occurre d primarily in the substrate binding regions to cope with an increasin g variety of foreign compounds. (C) 1994 Academic Press, Inc.