Properties As Enzyme

The main function of TGase is formation of e-(y-gluta-myl) lysine isopeptide crosslinks between naturally occurring proteins or peptides, resulting in polymerization (Figs. 1, 3). Amines such as hydroxylamine, mono-dansylcadaverine, cadaverine, spermine, spermidine, and putrescine can be incorporated into the protein substrate by the TGase reaction to bind covalently to glu-tamine residues resulting in the inhibition of the polymerization of proteins (Fig. 2). Thus, amines are competitive inhibitors for the TGase reaction, and can be utilized as one of substrates as described above. For example, there is a report that Km values of rat brain TGase for putrescine and N,N-dimethylcasein were 0.26 and 0.065 mM, respectively (52). The Km values of muscle TGases of some animals will be described later. In a case of the TGase from soybean (Glycine max) leaves, the Km values for putrescine, spermidine, and spermine were determined to be 0.109, 0.042, and 0.069 mM, respectively (53). Although this plant enzyme does not essentially require Ca2+, the Km values are similar to those of tissue TGases known in animals. Slime mold Physarum polycephalum TGase has Km values of 0.049, 0.021, and 0.032 mM for [14C]putrescine, [14C]spermidine, and [14C]spermine, respectively (54).

Figure 5 Molecular phylogenetic relationship among vertebrate transglutaminases. A tree was constructed by the neigbor-joining method (42) using the program MEGA (43). Bootstrap values > 50% are shown above or below branches. Filled diamond, open circle, open diamond, open square, and filled circles indicate blood-clotting Factor XIIIA, keratinocyte TGases, prostate gland TGases, erythrocyte band 4.2 (pallidin), and tissue TGases, respectively. The groups of them are considered to be paralogous to each other. The evolutionary distance is expressed in terms of amino acid substitutions per site. The tree is rooted with mesenchyme TGase of Ciona intestinalis embryo.

Figure 5 Molecular phylogenetic relationship among vertebrate transglutaminases. A tree was constructed by the neigbor-joining method (42) using the program MEGA (43). Bootstrap values > 50% are shown above or below branches. Filled diamond, open circle, open diamond, open square, and filled circles indicate blood-clotting Factor XIIIA, keratinocyte TGases, prostate gland TGases, erythrocyte band 4.2 (pallidin), and tissue TGases, respectively. The groups of them are considered to be paralogous to each other. The evolutionary distance is expressed in terms of amino acid substitutions per site. The tree is rooted with mesenchyme TGase of Ciona intestinalis embryo.

Inhibitors or cofactors were searched for mainly by an assay method utilizing amine incorporation. Metal ion chelating reagents such as EDTA and EGTA inhibit the activity of various TGases, which can be restored by the addition of Ca2+. In addition, iodoa-cetic acid, iodoacetamide, and p-chloromercuribenzo-ate inhibit TGase activity, while 2-mercaptoethanol and dithiothreitol (DTT) stabilize the activity. Therefore, TGase is well known as a Ca2+ -dependent SH enzyme.

On the other hand, some synthetic compounds such as 2-[3-(diallylamino)-propionyl]benzothiophene (55), and 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazo-nium chloride or L-68277 (56) are well-known non-competitive inhibitors of TGase.

Tissue TGase is inhibited by nucleotides. ATP inhibits the activity of rat liver TGase in a concentration-dependent way. Complete inhibition was obtained with 3 mM ATP. ADP inhibited the TGase activity similarly to ATP, but AMP had much less inhibition. There was no significant inhibition by adenosine and adenine. CTP possessed the same inhibitory activity as that of ATP, while GTP and UTP had - 50% of the ATP-induced inhibition (57). Such inhibitory effect of

ATP or GTP has been well elucidated for various tissue TGases [guinea pig liver TGase (5); erythrocyte TGase (59); Factor XIII (60); rat brain TGase (52)]. Moreover, it has been demonstrated that a human tissue TGase has nucleotide hydrolysis activity similar to ATPase or GTPase (61). This raises the possibility that the enzyme regulates cell receptor signaling (62). Studies show that the GTP and ATP hydrolysis sites are localized within the core domain of the tissue TGase (63). Recently, a 77-kDa GTP-binding protein, Gah5, was isolated from pig heart membranes with a binding affinity of nucleotides in order: GTP > GDP > ITP ยป ATP, which is similar to that observed for other G-proteins involved in receptor signaling. The Gah5 also exhibits TGase activity (64).

The optimum pH of the TGase activity was highly variable as follows: 7.5 for 101-kDa Physarum polyce-phalum TGase (65); 8.0 for Caenorhabditis elegans TGase (66); 9.0-9.5 for red sea bream TGase (67), and 5-6 for egg envelope TGases of rainbow trout (13-15).

For guinea pig liver TGase, the optimal incorporation of amines into proteins occurs at pH 7.2-8.5 (2). However, the pH of optimal ammonia liberation (see

Figure 6 Three-dimensional structure of human blood-clotting Factor XIIIA. The coordinate file for the human blood clotting factor XIIIA dimer [PDB Id., 1GGU (44)], which is supported by Brookhaven Protein Databank, was obtained from National Library of Medicine (NCBI), and the 3D structure was displayed by Molecular Visualization Program, RasMol ver 2.5 (128). The overall structure is characterized by the activation peptide (AP, arrow heads) and four domains, the ^-sandwich, the catalytic core, barrels 1 and 2 (44, 48). Due to unresolved problems, it appears that each monomer is missing the eight amino acid residues from the N-terminus (1-8), the linker between the activation peptides and ^-sandwich (30-43), the sequence between the core and barrel 1 (508-516), and the C-termini (728-731). (A) Overall structure of the Factor XIIIA dimer. Two monomer molecules are visible in parallel, symmetrically with respect to the center of the figure (+). The cysteine residue in the catalytic site (Cys314) is represented by a solid sphere (SH). (B) Left side view of A. The left and right chains in A are displayed in light and dark, respectively. Two spheres (Ca) are Ca2+ ions in both molecules.

Figure 6 Three-dimensional structure of human blood-clotting Factor XIIIA. The coordinate file for the human blood clotting factor XIIIA dimer [PDB Id., 1GGU (44)], which is supported by Brookhaven Protein Databank, was obtained from National Library of Medicine (NCBI), and the 3D structure was displayed by Molecular Visualization Program, RasMol ver 2.5 (128). The overall structure is characterized by the activation peptide (AP, arrow heads) and four domains, the ^-sandwich, the catalytic core, barrels 1 and 2 (44, 48). Due to unresolved problems, it appears that each monomer is missing the eight amino acid residues from the N-terminus (1-8), the linker between the activation peptides and ^-sandwich (30-43), the sequence between the core and barrel 1 (508-516), and the C-termini (728-731). (A) Overall structure of the Factor XIIIA dimer. Two monomer molecules are visible in parallel, symmetrically with respect to the center of the figure (+). The cysteine residue in the catalytic site (Cys314) is represented by a solid sphere (SH). (B) Left side view of A. The left and right chains in A are displayed in light and dark, respectively. Two spheres (Ca) are Ca2+ ions in both molecules.

Fig. 1) varies with the substrate: 7.0-7.5 for unmodified insulin; 6.5-7.0 for acetylated insulin (68). On the other hand, there is a report for the same enzyme that the optimal incorporation of hydroxylamine into N-carbobenzoxy (CBZ)-L-glutaminylglycine occurs near pH 6.0 (69).

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