Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of l -γ-glutamylamines producing 5-oxo- l -proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on l -γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between l -γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of l -γ-glutamylamines. The isodipeptide N ɛ -( l -γ-glutamyl)- l -lysine 1 was used as a reference. The kinetic constants of the l -γ-glutamyl derivative of n- butylamine 7 , were nearly identical to those of 1 . Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in l -γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on l -γ-glutamyl amino acids except for l -γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in l -γ-glutamylamines restored activity for gGACT, and l -γ-glutamylneohexylamine 19 had a higher specificity constant ( k cat /K m ) than 1 . gGACT did not exhibit any stereospecificity in the amide region of l -γ-glutamylamine substrates. In addition, analogues ( 26 – 30 ) with heteroatom substitutions for the γ methylene position of the l -γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of l -cysteine ( 28 – 30 ) were excellent substrates for gGACT.