J. Biol. Chem., Vol. 266, Issue 2, 830-836, Jan, 1991
Inhibition of rat liver glutathione S-transferase isoenzymes by peptides stabilized against degradation by gamma-glutamyl transpeptidase
AE Adang, J Brussee, A van der Gen and GJ Mulder
Division of Toxicology, University of Leiden, The Netherlands.
Inhibitors for glutathione S-transferase (GST) iso-enzymes from rat liver
with high affinity for the glutathione-binding site (G-site) have been
developed. In previous studies, a model was described for the G- site of
GST (Adang, A. E. P., Brussee, J., van der Gen, A., and Mulder, G. J.
(1990) Biochem. J. 269, 47-54) in terms of essential and nonessential
interactions between groups in glutathione (GSH) and the G- site. Based on
this model, compounds were designed that have high affinity for the G-site
but cannot be conjugated. In the dipeptide gamma-L-glutamyl-D-aminoadipic
acid (gamma-L-Glu-D-Aad), the L- cysteinylglycine moiety is replaced by
D-aminoadipic acid. This dipeptide is an efficient competitive inhibitor
(toward GSH) of mu class GST isoenzymes with Ki values of 34 microM for GST
isoenzyme 3-3 and 8 microM for GST isoenzyme 4-4. Other GSH-dependent
enzymes, such as gamma-glutamyl transpeptidase (gamma-GT), glutathione
reductase, and glutathione peroxidase, were not inhibited by 1 mM of
gamma-L-Glu-D- Aad. Inhibition is also highly stereospecific since
gamma-L-Glu-L-Aad is only a poor inhibitor (Ki = 430 microM for GST 3-3).
Gamma-L- Glutamyl-D-norleucine also had a much higher Ki value for GST 3-3.
Thus, the presence of a delta-carboxylate group in D-Aad appears to be
essential for a high affinity inhibitor. An additional hydrophobic group
did not result in increased inhibitory potency. In a different approach,
the gamma-L-glutamyl moiety in GSH was replaced by delta-L- aminoadipic
acid; delta-L-Aad-L-Cys-Gly is an efficient cosubstrate analogue for GSTs
with Km values comparable to GSH and Vmax values ranging from 0.24 to 57
mumol/min/mg for the different GSTs. The structures of the efficient
inhibitor and the cosubstrate analogue were combined in delta-L-Aad-D-Aad,
which had a Ki value of 68 microM with GST 3-3. In order to investigate
their possible use in vivo studies, the degradation of gamma-L-Glu-D-Aad
and delta-L-Aad-L-Cys-Gly by gamma- GT was investigated. The peptides
showed no measurable hydrolysis rates under conditions where GSH was
rapidly hydrolyzed. Thus, an efficient, mu class-specific GST inhibitor and
a gamma-glutamyl-modified cosubstrate analogue of GSH were developed. Their
gamma-GT stability offers the possibility to use these peptides in in vivo
experiments.
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