ALDEHYDE AND PHOSPHINATE ANALOGS OF GLUTATHIONE AND GLUTATHIONYLSPERMIDINE - POTENT, SELECTIVE BINDING INHIBITORS OF THE ESCHERICHIA-COLI BIFUNCTIONAL GLUTATHIONYLSPERMIDINE SYNTHETASE AMIDASE/

Introduction: The tripeptide glutathione is converted to glutathionyls permidine (Gsp) in Escherichia coli and in trypanosomatid parasites by an ATP-cleaving Gsp synthetase activity. In parasites, an additional glutathionylation forms bis-(glutathionyl)-spermidine, trypanothione, believed to be the major surveillance thiol involved in oxidant defens e mechanisms in trypanosomatid parasites. In E. coli, the Gsp syntheta se is part of a bifunctional enzyme opposed by the hydrolytic Gsp amid ase. Results: Gsp amidase and Gsp synthetase activities of the bifunct ional E. coli enzyme can be separately targeted by potent, selective s low-binding inhibitors that induce time-dependent inhibition. The inhi bitor gamma-Glu-Ala-Gly.CHO most probably captures Cys59 and accumulat es as the tetrahedral adduct in the amidase active site. Inhibitory Gs p phosphinate analogs are phosphorylated by ATP to yield phosphinophos phate analogs in the synthetase active site. Binding of phosphinophosp hate in the Gsp synthetase active site potentiates the inhibition affi nity for the aldehyde at the Gsp amidase active site by two orders of magnitude, Conclusions: Time-dependent inhibition of the Gsp amidase a ctivity by the aldehyde substrate analog supports previous work that s uggests glutathionyl acyl-enzyme intermediate formation in the Gsp ami dase reaction mechanism. Use of potent selective inhibitors against Gs p amidase (aldehyde) and Gsp synthetase (phosphinate) activities provi des further evidence of interdomain communication in the bifunctional enzyme from E. coli.