Interaction of gamma-glutamyl transpeptidase with acivicin.
Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.
Stole
E
E
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Smith
T K
TK
Manning
J M
JM
Meister
A
A
D000818:Animals; D002384:Catalysis; D004793:Enzyme Reactivators / Q000494:pharmacology; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology*; D007555:Isoxazoles / Q000378:metabolism*; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism*
D004793:Enzyme Reactivators; D005971:Glutamates; D006898:Hydroxylamines; D007555:Isoxazoles; D019811:Hydroxylamine; D018698:Glutamic Acid; D005723:gamma-Glutamyltransferase; C006303:acivicin