Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity

Abstract

T ransglutaminase 2 [here designated TG2 (EC2.3.2.13), but also identified as the GTP-binding protein, G h ] differs from its relatives in the family of Ca 2ϩ -dependent protein crosslinking enzymes mainly by its affinity for nucleotides (1-3) and for fibronectin (4-8). GTP-binding and hydrolysis enable TG2 to act as a G protein in signal transduction (G h ; ref. 9); GTP also serves as a potent allosteric inhibitor that suppresses the Ca 2ϩ -activated crosslinking activities of the enzyme. Independently of any of its catalytic functions and GTP-binding activity, TG2 binds fibronectin with very high affinity (7). Thus, when expressed on cell surfaces, TG2 plays an important role in organizing the extracellular matrix as an integrin-binding adhesion coreceptor (10, 11).Crystal structures are available for two related proteins: the A subunit of the coagulation factor XIII zymogen (fXIIIA; ref. 12) and the sea bream liver TG (13). Although neither of these is regulated by GTP (personal information from Kohki Ishikawa regarding the latter enzyme), sequence homologies between TG2 (rat) and these proteins suggest that TG2͞G h also would be organized into four domains: a ␤ sandwich (residues 1-138) followed by a large ␣͞␤ catalytic core (139-471) and two ␤ barrels [barrel 1 (472-584) and barrel 2 (585-686)]. The N-terminal (Ϸ28 kDa) region of TG2 is responsible for binding fibronectin (8) whereas the C-terminal segment of barrel 2 could interact with phospholipase in signal transduction (14). The core domain comprises the papain-like catalytic center (15) and the nucleotidebinding residues (16). Like the monomeric but unlike the heterotrimeric G proteins, TG2 was shown to bind 2Ј-(or 3Ј)-O-(Nmethylanthraniloyl)GTP (mantGTP) with high affinity (17). Binding was evidenced also by the appearance of an energy transfer band from one or more Trp residues of the protein to the mant fluorophore in this nucleotide. Because the core domain contains almost all of the tryptophans conserved throughout the TG family, we evaluated the nucleotide-binding properties and the transamidating activities of candidate tryptophan point mutants of TG2. Specifically, W180, 241, 278, 332, and 337 were targeted because they surround S171, a residue identified to be critical for GTPbinding. On a model of TG2 based on the fXIIIA zymogen crystal structure (16), all of the mutated residues are within 20Å (range 9.3-18.7Å) of both S171 and the active-site cysteine C277. We show that no single Trp residue alone accounts for the observed energy transfer from the protein to the nucleotide but some of the tryptophan mutants displayed greatly diminished mantGTPbinding affinities. Another important finding was that residue W241-that is highly conserved among the TGs but is lacking in the catalytically inactive family member of human red cell, band 4.2-is critical for transamidation by TG2. Materials and MethodsConstructs. Site-directed mutants of rat TG2͞G h cDNA were generated in a temperature-cycling reaction with Pfu DNA polymerase by using two complement...

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

“…In earlier studies with rat TG2 only changing Trp 241 resulted in significant decrease of transamidase activity (Murthy et al 2002). In case of human TG2 not only the hydrophobicity of the amino acids around the active site and in the substrate binding area plays a role during the reaction catalysis, but the complexity of the surrounding Trp-s (space filling property, aromatic carbon atom ring containing nitrogen) also have important effect on stabilisation of the transition states of transglutaminase activities (recently reviewed by Keillor et al 2014).…”

“…Comparing the known crystal structures of human transglutaminases with corresponding papain structures reveals isolation of the active site of both from ambient water by being buried in the core domain, and surrounded by bulky hydrophobic residues (Pinkas et al 2007, Chica et al 2004, Iismaa et al 2003, Murthy et al 2002. Although space-filling models in the presence of substrate (inhibitor) show the active site quite exposed to water, in the absence of substrate the water is repelled from the catalytic cavity by hydrophobic tunnels isolated by annealing hydrophobic side chains, such as W241-W332, W278-F334.…”

“…Based on this consideration we designed 12 single mutants namely: W180F, W180L, W241F, W241L, W278F, W278L, W332F, W332L, F334L, W337F, W337L, Y516L. These mutants in human TG2 have not yet been purposefully created for the characterisation of their isopeptidase property (Murthy et al 2002, Iismaa et al 2003.…”

“…In case of human TG2 not only the hydrophobicity of the amino acids around the active site and in the substrate binding area plays a role during the reaction catalysis, but the complexity of the surrounding Trp-s (space filling property, aromatic carbon atom ring containing nitrogen) also have important effect on stabilisation of the transition states of transglutaminase activities (recently reviewed by Keillor et al 2014). Pinkas et al (2007) reported that the W332A mutation in human TG2 and the W332F mutation in rat TG2 lead to loss or lower transamidase activity, respectively (Murthy et al 2002), but in these articles only the transamidase activity was tested. The demonstrated functional difference between orthologous TG2 enzymes also makes comparison difficult (Ruan et al 2008).…”

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2

“…However, this mutation results in a conformational change that impairs GTP binding capability [23]. A conserved tryptophan residue (Trp241) is also crucial for the transamidation activity of the protein [24], and mutation of this residue to phenylalanine or alanine abolishes crosslinking activity, however it does not diminish guanine nucleotide binding [25].…”