A model for the reaction mechanism of the transglutaminase 3 enzyme

Abstract

A bstractTransglutam inase enzym es (TG ases) catalyze the calcium dependent formation of an isopeptide bond betw een protein-bound glutam ine and lysine substrates. Previously w e have show n that activated TG ase 3 acquires tw o additional calcium ions at site tw o and three. The calcium ion at site three results in the opening of a channel. A t this site, the channel opening and closing could m odulate, depending on which m etal is bound. Here we propose that the front of the channel could be used by the tw o substrates for enzym e reaction. W e propose that the glutam ine substrate is directed from Trp236 into the enzym e, shown by m olecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to form ation of the isopeptide bond. Further, direct com parisons of the structures of TG ase 3 w ith other TG ases have allow ed us to identify several residues that m ight potentially be involved in generic and specific recognition of the glutam ine and lysine substrates.Keywords: calcium ions; protein structure; residue specificity; TGase; TGase mechanism; X-ray crystallography IntroductionTransglutaminases (TGase; protein-glutamine: amine γ-glutamyl-transferase) are a family of calcium-dependent acyl-transfer enzymes that are widely expressed in biota (Folk and Chung, 1985;Greenberg et al., 1991;Melino et al., 1998;Melino et al., 2000;Fesus and Piacentini, 2002). Each of the eight active human TGase enzyme isoforms can activate a protein-bound Gln residue to form a thiol-acyl enzyme intermediate, which is attacked by a second nucleophilic substrate to accomplish the two-step reaction. The reaction leads to the formation of an isopeptide bond between two proteins and the covalent incorporation of polyamine into protein (Folk and Chung, 1985;Greenberg et al., 1991). The nucleophile may be: water, so that the activated Gln residue is deamidated to a Glu; a polyamine, so that a mono-substituted adduct or bi-substituted cross-link is formed; an alcohol, particularly the ω-hydroxyl group of certain long-chain ceramides expressed in mammalian epidermis, to form an ester; and perhaps most commonly, an ε-NH2 group of a protein bound Lys residue, resulting in the formation of an N ε -(γ-glutamyl)lysine isopeptide cross-link Nemes et al., 2000). This stabilizes macromolecular protein complexes. Typically, TGases recognize the generic sequence motif Gln-Gln*-Val for the first step of the reaction (where Gln* represents the targeted residue), although different isoforms display specificity as to which substrates bearing the motif may approach the enzyme . Anecdotal data suggest less specificity for Lys substrates (Tarcsa et al., 1998;Candi et al., 1999;.The three dimensional structures of four TGases have now been reported, i.e. human factor XIIIa (hfXIIIa) ), TGase 2 (Liu et al., 2002, TGase 3 enzymes (Ahvazi et al., 2002;2003), and a fish enzyme (fTG, equivalent to mammalian TGase 2, Noguchi et al., 2001). All consist of four domains that are similar in organization and fold (Figure 1...

Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis

“…maintained in low (0.09 mM) calcium-containing medium. This is surprising, because calcium is required for transglutaminase activity (89,90). Our present and past (25,26) findings suggest that TG1 can be activated without an increase in intracellular calcium.…”

Structural Basis for the Coordinated Regulation of Transglutaminase 3 by Guanine Nucleotides and Calcium/Magnesium

“…Moreover, we have shown that an increase in Mg 2ϩ ion concentrations alone, even without GTP, can inhibit the enzyme, raising the possibility that GTP binding and Mg 2ϩ ion substitution at site 3 represent a coordinated event in the inactivation of TGase 3 (28). A set of concerted changes in the enzyme structure, stemming from the binding of GTP and Mg 2ϩ ion, cause the closing of the channel required for substrate access to the active site (47). Thus, the GTP␥S imparts the inactive state through a set of local structural rearrangements and not through its direct occlusion of the active site.…”

“…Furthermore, it has been proposed that the front of the channel could be used by the two substrates for enzyme reaction (47), based on detailed analyses of current structural information on the zymogen and the activated forms of TGase 3. Therefore, we examined the crystal structures in the presence of GTP␥S versus GDP in terms of their implications for substrate access to the active site residues including Cys 272 .…”

Perturbations in protein dynamics associated with ligand binding to the blood coagulation enzymes thrombin and Factor XIII.

“…Whereas the γ′ peptide-IIa interface has been localized to ABE-II, the binding site for GpIbα is more ambiguous and may be at ABE-II or ABE-I. The C-terminal tail of GpIbα (269)(270)(271)(272)(273)(274)(275)(276)(277)(278)(279)(280)(281)(282)(283)(284)(285)(286) shares similar sequence elements with the γ′ peptide. Both GpIbα X-ray structures (96,97) display comparable ABE-II interactions for the C-terminal tail; however, the Celikel structure (1OOK) shows the C-terminal tail dimerizing thrombin through binding with ABE-I in a symmetry related thrombin molecule (97).…”

“…At the present time, a peptide-based anticoagulant that targets ABE-II has not been developed. By examining the conformational features associated with γ′ peptide (410-427) and GpIbα (269)(270)(271)(272)(273)(274)(275)(276)(277)(278)(279)(280)(281)(282)(283)(284)(285)(286) binding to ABE-II, an ABE-II targeting peptide with anticoagulant properties may be developed employing the unique structural elements characteristic of these complexes.…”