Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Abstract

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant Km (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant kcat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transition-state stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower kcat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transition-state-stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Fig. 1.

Stereo image showing superposition of active sites of TG2 (Cys-277, His-335, Asp-358; blue) and the papain (Cys-25, His-159, Asn-175; red)–leupeptin (yellow) complex. Distances (dotted lines) between the leupeptin oxyanion and the side-chain amide nitrogen of Gln-19 (red) of papain, or of Trp-241 (blue) of TG2, are 2.9 and 3.4 Å, respectively. Distances between the leupeptin oxyanion and the backbone amide nitrogen of Cys-25 of papain, or of Cys-277 of TG2, are 3.0 and 2.9 Å, respectively.

Fig. 2.

Partial alignment (29) of human TGs, TGs representative of major branches of the TG phylogenetic tree (1), and papain. Active site Cys, His, and Asp (Asn in papain) are highlighted in red (active-site numbering is for TG2). Trp-241 equivalent of TG2 and transition-state-stabilizing Gln-19 of papain are highlighted in purple.

Fig. 3.

Verification of components of the DC–β-casein_161–175 conjugate assay for TG activity. (A) HPLC elution and spectral analysis (Inset) of TG assay substrates (DC and β-casein_161–175 peptide) and the DC–β-casein_161–175-conjugated product. WT TG2 (10 pmol) was incubated for 60 min with 0.5 mM DC and 0.75 mM β-casein_161–175. HPLC elution was monitored at 214 nm (green trace) and 320 nm (black trace). (Inset) DC (blue dotted line), β-casein_161–175 (red dotted line), and DC–β-casein_161–175 conjugate (solid black line). (B and C) MALDI-TOF MS analysis (linear mode) of β-casein_161–175 and DC–β-casein_161–175, respectively.

Fig. 4.

GTP photolabeling of WT and Trp-241 mutant proteins (0.2–2.0 μg). Gels were visualized by Coomassie blue staining (A) and autoradiographed for 7 days at –80°C (B). After densitometry, GTP-labeling intensity was normalized for amount of protein. Labeling efficiencies for mutant proteins, relative to WT TG2, were 1.4 ± 0.06 for W241Y, 1.1 ± 0.06 for W241F, 2.1 ± 0.5 for W241H, 1.4 ± 0.3 for W241G, and 1.4 ± 0.5 for W241A.

Fig. 5.

Reaction pathway and a proposed mechanism for TG2-catalyzed transamidations, based on the papain-reaction mechanism. Active-site Cys-277 and His-335 form a thiolate–imidazolium ion pair. Binding of the Gln-containing substrate 1 enables the thiolate to attack the γ-carboxamide group in the substrate. The reaction proceeds through oxyanion intermediate 1 that is stabilized by H-bonding both to the backbone nitrogen of Cys-277 and to the Nε1 nitrogen of Trp-241. In the ensuing acylation step, NH₃ is released as acylenzyme intermediate is formed. A distinguishing feature of TGs, which sets these enzymes apart from papain proteases, is a high specificity for the second substrate manifested in a saturable complex between substrate 2 and the acylenzyme intermediate. Nucleophilic attack by the amino group of substrate 2 leads to formation of oxyanion intermediate 2 that is again stabilized by H-bonding interactions to both Cys-277 and Trp-241. In the final deacylation step, cross-linked product is released and TG2 is regenerated.

Scheme 1.