Identification and Characterization of γ-Glutamylamine Cyclotransferase, an Enzyme Responsible for γ-Glutamyl-ϵ-lysine Catabolism

Abstract

␥-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts ␥-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward ␥-glutamyl-⑀-lysine, derived from the breakdown of fibrin and other proteins crosslinked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in ␥-glutamylcyclotransferase (GGCT), an enzyme with activity toward ␥-glutamyl-␣-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu 82 ). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.Proteins can be cross-linked via the side chains of glutamine and lysine by transglutaminases. This reaction results in the formation of ammonia and an L-␥-glutamyl-L-⑀-lysine isopeptide bond linking the two polypeptide chains. The existence of such ␥-glutamyl-⑀-lysine links was unambiguously demonstrated in factor XIIIa-cross-linked fibrin: extensive proteolytic degradation of cross-linked fibrin resulted in the formation of L-␥-glutamyl-L-⑀-lysine (1). It was concluded that L-␥-glutamyl-L-⑀-lysine is not broken down by conventional proteolysis. The breakdown of the isodipeptide is instead catalyzed by ␥-glutamylamine cyclotransferase (GGACT), 2 first purified from rabbit kidney (2, 3). The partially purified enzyme was demonstrated to be active toward a range of L-␥-glutamyl conjugates with mono-and polyamines and amino acids. The action of GGACT in all cases is to cyclize the ␥-glutamyl moiety, producing 5-oxo-L-proline and the free alkylamine. No activity was detected toward L-glutamine or L-␤-aspartyl-L-⑀-lysine. Furthermore, derivatives of L-␥-glutamyl-L-⑀-lysine in which the ␣-amino or ␣-carboxyl functional group of the glutamyl moiety is blocked do not serve as substrates, nor do any of a range of L-␥-glutamyl-L-␣-amino acids (3). Based on these results, it was proposed that GGACT functions in the latter stages of the catabolism of the products of transglutaminases (3).We reported recently the identification, cloning, and threedimensional structure of an enzyme with related catalytic activity to GGACT but with distinct specificity: ␥-glutamylcyclotransferase (GGCT) (4). Unlike GGACT, this enzyme is active toward a range of L-␥-glutamyl-␣-amino acids (5, 6). GGCT catalyzes the penultimate step in glutathione catabolism, whereby L-␥-glutamyl-L-␣-cysteine and other L-␥-glutamyl-L-␣-amino acid dipeptides formed by the ␥-glutamyl cycle are c...

Emerging Regulatory Paradigms in Glutathione Metabolism

“…Although GGCT was first purified nearly 40 years ago (Board et al, 1978; Orlowski et al, 1969; Palekar et al, 1974), the gene encoding this enzyme has only recently been identified (Oakley et al, 2008). It shares sequence and structural similarity to γ-glutamylamine cyclotransferase, which degrades γ-glutamyl-ɛ-lysine liberated from proteins covalently crosslinked by transglutaminases (Oakley et al, 2010). Furthermore, this enzyme family is broadly represented in bacteria, plants, and other higher eukaryotes.…”

Structure and Function of Allophanate Hydrolase

“…S1). A search with the Dali server indicated that this structure is homologous to the structures of ␥-glutamyl cyclotransferase (40) and ␥-glutamylamine cyclotransferase (GGACT) (41), and a number of proteins without known functions (Protein Data Bank codes 2QIK, 1V30, 2KL2, 2G0Q, 2I5T, 2Q53, 1XHS, and 2JQV). After alignment, the r.m.s.…”

“…At the GGACT active site, hydrogen bonds are formed between the 5-oxo-L-proline carboxyl and the main chain amides of Tyr 7 and Gly 8 ( Fig. 7C) (41). Similarities between the active sites of GGACT and the C domain prompted us to model N-carboxycarbamate into the C domain active site with one of its carboxyl groups forming similar hydrogen bonds with main chain amides of Val 489 and Gly 490 .…”

Synthesis and GGCT Inhibitory Activity of <i>N</i>-Glutaryl-L-alanine Analogues

“…After the typical procedure of amino-bond formation for (R)-benzyl 3-(N-tert-butoxycarbonyl) aminoglutaric acid with L-alanine benzyl ester, the crude product was dissolved in CH 2 Cl 2 (7 mL) and reacted with TFA (3 mL) for 10 h at r.t. Solvent was removed and the crude product was purified by column chromatography on silica gel eluted with 20% MeOH in CHCl 3 …”

“…Glutaryl-L-alanine (2) showed 87% inhibition of GGCT activity (entry 1). In comparison with 2, compounds with one less and one additional carbon, N-succinyl-L-alanine (3) and N-adipinyl-L-alanine (4), were poorly effective (entries 2, 3). A methyl ester at the alanine unit (5) showed 22% inhibition (entry 4) and a methyl ester at the glutaryl unit (6) exhibited 41% of activity (entry 5).…”