Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis

Abstract

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

“…The latter is in contrast with TvTIM1-Ile45Ala mutant that behaves a monomeric protein in comparison to the wild-type TvTIM1 ( Fig. 9 )[26]. An enzymatic characterization of LvTIM-Cys47Ala indicates that its calculated catalytic efficiency ( k cat /K m ) was 1.4 × 10 5 min −1 mM −1 , this catalytic efficiency is similar to the one observed for the wild-type LvTIM.…”

“…The amino acid character of the "ball" is usually an Ala, Val, Ile or Thr and the socket is not conserved, although Phe, Ile or Met are generally present ( Fig. 7 panel B ) [26]. Considering the properties of cysteine (similar side chain length as an Ala), the prediction of Lara et al [26] is that Cys 47 favors the monomeric state in TIMs The observed structural stability between LvTIM and TvTIMs correlates with the hydrophobicity of the ball : TvTIM1-Ile45 > TvTIM2-Val45 > LvTIM-Cys47.…”

“…To understand if the “ball and socket” interplay is necessary for dimer formation in LvTIM, we constructed a mutant in which cysteine 47 was replaced by an alanine (LvTIM-Cys47Ala), this mutant eliminated the thiol group and reduced the accessible surface area (ASA) by 37 Å 2 . In the case of TvTIM2 the substitution of Valine 45 to Alanine decrease the ASA by 50 Å 2 and completely alters the equilibrium from dimer to monomer [16, 26]. A gel filtration experiment of purified LvTIM-Cys47Ala with wild-type LvTIM shows that both proteins are dimers at concentrations of 2 and 0.2 mg ml −1 .…”

“…The exceptions to this fact are TIMs from Trichomonas vaginalis (TvTIMs). These TIMs only differ between them in 4 among 252 amino acids and the energy necessary to dissociate their dimers is lower than the energy to unfold its monomers [16, 25, 26]. In order to understand glycolysis regulation in crustaceans we characterized the triosephosphate isomerase from shrimp Litopenaeus vannamei (LvTIM) with focusing on its stability and the "ball and socket" model postulated for TvTIMs [16, 26].…”

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

“…1C). The amino acid sequences of the two TvTIM proteins are 98.4% identical, differing by only four amino acids but showing remarkable differences in physicochemical properties, dimer stability, and enzymatic activity (23,24,44). Thus, it was impossible to differentiate between TvTIM1 and TvTIM2 using an anti-TvTIMr antibody.…”