Dissection of the catalytic mechanism of isozyme 4-4 of glutathione S-transferase with alternative substrates.

The kinetic and chemical mechanism of isozyme 4-4 of rat liver glutathione (GSH) S-transferase was investigated by using several alternative peptide substrates including N-acetyl-GSH, gamma-L-glutamyl-L-cysteine (gamma-GluCys), N4-(malonyl-D-cysteinyl)-L-2,4-diaminobutyrate (retro-GSH), and N4-(N-acetyl-D-cysteinyl)-L-2,4-diaminobutyrate (decarboxylated retro-GSH). The enzyme, which is normally stereospecific in the addition of GSH to the oxirane carbon of R absolute configuration in arene oxide substrates, loses its stereospecificity toward phenanthrene 9,10-oxide with the retro peptide analogues, giving a 2:1 mixture of the S,S and R,R stereoisomeric 9,10-dihydro-9-(S-peptidyl)-10-hydroxyphenanthrenes. The analogues with normal peptide bonds (N-acetyl-GSH and gamma-GluCys) show normal stereospecific addition. The kinetic mechanism of the enzyme was investigated by using the alternative substrate diagnostic with several 4-substituted 1-chloro-2-nitrobenzenes and GSH, N-acetyl-GSH, and gamma-GluCys. Varying the concentration of electrophile vs the identity of the GSH analogue and the concentration of GSH vs the identity of the electrophile gave two sets of intersecting reciprocal plots, a result consistent with a random sequential kinetic mechanism. The pH profiles of kc and kc/Ksm [saturating GSH, variable 1-chloro-2,4-dinitrobenzene (1)] exhibit a dependence on a deprotonation in the enzyme-GSH-1 and enzyme-GSH complexes with molecular pKa's of 6.1 and 6.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)