Gamma-glutamylamine Cyclotransferase Research Articles

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(ε)-(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.

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

Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-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.

Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.

Aspartyl- and Glutamyl-Lysine Crosslinks Formation and Their Nutritional Availability

Transglutaminase-catalyzed incorporation of L-lysine into wheat gliadin rendered the lysine-fortified protein poorly digestible in the in vitro tests. In rat feeding tests, however, the luminal leavings and excreta collected after administration of the [14C]lysine-fortified gliadin contained less than one-tenth of the radioactivity originally administered to rats. The enzymes, gamma-glutamylamine cyclotransferase and 5-oxoprolinase, known to occur in animal kidney are at least in part responsible for the observed high availability of isopeptide bound lysine. A novel enzyme which is capable of directly hydrolyzing the cross-linked isopeptide into component amino acids and peptides, "N epsilon-(gamma-glutamyl)lysine hydrolase" was found in the isolated microorganisms which can use the synthesized N epsilon-(gamma-glutamyl)lysine as their only source of carbon and nitrogen. The enzyme(s) appear to be effectively used for improving digestibility and availability of protein matrixes formed in normal metabolism and by heat and/or shear treatment commonly used in food processing.

Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.

Activation of transglutaminase and production of protein-bound gamma-glutamylhistamine in stimulated mouse mast cells.

The identification of transglutaminase in the growth-factor-dependent mouse mast cell line PT18 was accomplished through its characteristic catalytic properties (specificity, calcium dependency, and inhibition by iodoacetamide); and by both immunoprecipitation and Western blot analysis using affinity purified antibody. The enzymatic activity in these cells increased in association with the release of histamine from the cells induced by an IgE-dependent mechanism or by exposure to the ionophores A23187 or Br-x537A. The increase in transglutaminase activity was paralleled by a marked increase in the level of protein-bound gamma-glutamylhistamine, determined in radiolabeled form in mast cells that were either metabolically labeled with [3H]histidine or incubated with [3H]histamine before degranulation. The highest level of bound gamma-glutamylhistamine was found in the immunologically stimulated cells. Enzymatic activity and the gamma-glutamyl derivative were associated primarily with the cells, both before and after stimulation. Separation of gamma-glutamylhistamine in a proteolytic digest of these cells was carried out using a combination of ion exchange chromatography and high performance liquid chromatography. The gamma-glutamyl compound was identified and quantitated through the enzymatic production of histamine with the use of gamma-glutamylamine cyclotransferase, an enzyme specific for the disassembly of gamma-glutamylamines.

Gamma-Glutamylamine cyclotransferase. An enzyme involved in the catabolism of epsilon-(gamma-glutamyl)lysine and other gamma-glutamylamines.

gamma-Glutamylamine cyclotransferase, an enzyme found in a number of animal tissues and cells, catalyzes the conversion of epsilon-(L-gamma-glutamyl)-L-lysine to free lysine and 5-oxo-L-proline as well as the release of free amines and the formation of 5-oxo-L-proline from a variety of other L-gamma-glutamylamines. Among its substrates are both the mon- and di-gamma-glutamyl derivatives of putrescine, spermidine and spermine, and a derivative of epsilon-(L-gamma-glutamyl)-L-lysine in which both the alpha-amino group and the carboxyl group of the lysine moiety are blocked. The enzyme does not act on most gamma-glutamyl-alpha-amino acids, nor is it active toward the epsilon-lysyl derivatives of L-aspartic acid or D-glutamic acid. Derivatives of epsilon-(L-gamma-glutamyl)-L-lysine in which the alpha-amino or the alpha-carboxyl function of the glutamyl moiety is blocked also do not serve as substrates. The specificity of gamma-glutamylamine cyclotransferase is in accordance with the proposal that it functions biologically in the latter stages of the catabolism of products of the action of transglutaminases. Some suggestions as to the manner in which gamma-glutamylamine cyclotransferase serves this function are made based on present knowledge of protein degradation.

Gamma-Glutamylamine cyclotransferase: specificity toward epsilon-(L-gamma-glutamyl)-L-lysine and related compounds.

gamma-Glutamylamine cyclotransferase, an enzyme that catalyzes the conversion of L-gamma-glutamylamines to free amines and 5-oxo-L-proline, was found in rabbit kidney and in various other tissues. The specificity of this enzyme indicates that it functions in the metabolism of products of transglutaminase action; among its substrates are epsilon-(L-gamma-glutamyl)-L-lysine, a derivative of this peptide in which the alpha-amino and carboxyl groups of the lysine moiety are blocked, and L-gamma-glutamyl-polyamine derivatives.