DESIGN, SYNTHESIS, AND BIOCHEMICAL EVALUATION OF PHOSPHONATE AND PHOSPHONAMIDATE ANALOGS OF GLUTATHIONYLSPERMIDINE AS INHIBITORS OF GLUTATHIONYLSPERMIDINE SYNTHETASE AMIDASE FROM ESCHERICHIA-COLI/

Three phosphapeptides designed to mimic two distinct tetrahedral inter mediates formed during either the synthesis or hydrolysis of glutathio nylspermidine (Gsp) were synthesized and evaluated as inhibitors of th e bifunctional enzyme Gsp synthetase/amidase. While the polyamine-cont aining phosphapeptides were determined to be potent and selective inhi bitors, they selectively inhibit the synthetase activity over the amid ase domain. A phosphonate-containing tetrahedral mimic is a reversible mixed-type inhibitor of Gsp synthetase with an inhibition constant of 6 mu M for the inhibitor binding to the free enzyme (K-i) and 14 mu M for the inhibitor binding to the enzyme-substrate complex (K-i'). The corresponding phosphonamidate is a slow-binding inhibitor with a K-i of 24 mu M and a K-i (isomerization inhibition constant) of 0.88 mu M A non-polyamine-containing phosphonamidate exhibits no significant in hibition of the synthetase or amidase activity.