Stereoselective Catalysis of a Retro-Michael Reaction by Class Mu Glutathione Transferases. Consequences for the Internal Distribution of Products in the Active Site

Abstract

The reaction of glutathione (GSH) with trans-4-phenyl-3-buten-2-one (PBO) is readily reversible in aqueous solution with an apparent (pH-dependent) equilibrium constant at pH 8 of 6.4 x 10(2) M-1. Two class mu isoenzymes of GSH transferase from rat (M1-1 and M2-2) and two site specific mutants (M1-1/V9I and M2-2/I9V) catalyze the addition of GSH to PBO and the elimination of GSH from the two diastereomeric products (isomers A and B) of 4-(S-glutathionyl)-4-phenyl-2-butanone with varying degrees of efficiency and stereoselectivity, with the major kinetic product in the addition reaction (isomer A) being the preferred substrate for the elimination reaction. The kinetic stereoselectivity of the addition reaction and the steady-state kinetics of the elimination reactions with product isomers A and B are used to estimate internal stereochemical equilibrium constants in which product isomer B is predominant. This result is consistent with the internal equilibrium constants measured under conditions of enzyme in excess. The results can be used to construct reaction coordinate diagrams for the interconversion of central complexes in the enzyme-catalyzed reactions. The possible metabolic consequences of the reversibility of additions of GSH to alpha, beta-unsaturated carbonyl compounds are discussed.