Dataset Information

The critical role of the loops of triosephosphate isomerase for its oligomerization, dynamics, and functionality.

ABSTRACT: Triosephosphate isomerase (TIM) catalyzes the reaction to convert dihydroxyacetone phosphate into glyceraldehyde 3-phosphate, and vice versa. In most organisms, its functional oligomeric state is a homodimer; however, tetramer formation in hyperthermophiles is required for functional activity. The tetrameric TIM structure also provides added stability to the structure, enabling it to function at more extreme temperatures. We apply Principal Component Analysis to find that the TIM structure space is clearly divided into two groups--the open and the closed TIM structures. The distribution of the structures in the open set is much sparser than that in the closed set, showing a greater conformational diversity of the open structures. We also apply the Elastic Network Model to four different TIM structures--an engineered monomeric structure, a dimeric structure from a mesophile--Trypanosoma brucei, and two tetrameric structures from hyperthermophiles Thermotoga maritima and Pyrococcus woesei. We find that dimerization not only stabilizes the structures, it also enhances their functional dynamics. Moreover, tetramerization of the hyperthermophilic structures increases their functional loop dynamics, enabling them to function in the destabilizing environment of extreme temperatures. Computations also show that the functional loop motions, especially loops 6 and 7, are highly coordinated. In summary, our computations reveal the underlying mechanism of the allosteric regulation of the functional loops of the TIM structures, and show that tetramerization of the structure as found in the hyperthermophilic organisms is required to maintain the coordination of the functional loops at a level similar to that in the dimeric mesophilic structure.

SUBMITTER: Katebi AR

PROVIDER: S-EPMC3926746 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The critical role of the loops of triosephosphate isomerase for its oligomerization, dynamics, and functionality.

Katebi Ataur R AR Jernigan Robert L RL

Protein science : a publication of the Protein Society 20131231 2

Triosephosphate isomerase (TIM) catalyzes the reaction to convert dihydroxyacetone phosphate into glyceraldehyde 3-phosphate, and vice versa. In most organisms, its functional oligomeric state is a homodimer; however, tetramer formation in hyperthermophiles is required for functional activity. The tetrameric TIM structure also provides added stability to the structure, enabling it to function at more extreme temperatures. We apply Principal Component Analysis to find that the TIM structure space ...[more]

PMID: 24318986

Similar Datasets

Project description:We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a approximately 50-fold increase in K(m) for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K(i) for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k(cat) for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 x 10(5)-fold decrease in k(cat)/K(m), corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D(2)O were quantified as dihydroxyacetone phosphate (DHAP) (27%), [1(R)-(2)H]DHAP (23%), [2(R)-(2)H]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D(2)O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >or=14-fold decrease in k(cat)/K(m) for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-(13)C]GA in D(2)O is [1-(13)C,2,2-di-(2)H]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k(cat)/K(m) for the reactions of both GAP and the neutral truncated substrate [1-(13)C]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".

Project description:Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the ?3, ?6, ?7 y ?8 of the TIM barrel. From these, the ?3-strand region (residues 59-66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-? structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where ?-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting ?-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway.

Project description:The role of the hydrophobic side chains of Ile-172 and Leu-232 in catalysis of the reversible isomerization of R-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TIM) from Trypanosoma brucei brucei (Tbb) has been investigated. The I172A and L232A mutations result in 100- and 6-fold decreases in k(cat)/K(m) for the isomerization reaction, respectively. The effect of the mutations on the product distributions for the catalyzed reactions of GAP and of [1-(13)C]-glycolaldehyde ([1-(13)C]-GA) in D(2)O is reported. The 40% yield of DHAP from wild-type Tbb TIM-catalyzed isomerization of GAP with intramolecular transfer of hydrogen is found to decrease to 13% and to 4%, respectively, for the reactions catalyzed by the I172A and L232A mutants. Likewise, the 13% yield of [2-(13)C]-GA from isomerization of [1-(13)C]-GA in D(2)O is found to decrease to 2% and to 1%, respectively, for the reactions catalyzed by the I172A and L232A mutants. The decrease in the yield of the product of intramolecular transfer of hydrogen is consistent with a repositioning of groups at the active site that favors transfer of the substrate-derived hydrogen to the protein or the oxygen anion of the bound intermediate. The I172A and L232A mutations result in (a) a >10-fold decrease (I172A) and a 17-fold increase (L232A) in the second-order rate constant for the TIM-catalyzed reaction of [1-(13)C]-GA in D(2)O, (b) a 170-fold decrease (I172A) and 25-fold increase (L232A) in the third-order rate constant for phosphite dianion (HPO(3)(2-)) activation of the TIM-catalyzed reaction of GA in D(2)O, and (c) a 1.5-fold decrease (I172A) and a larger 16-fold decrease (L232A) in K(d) for activation of TIM by HPO(3)(2-) in D(2)O. The effects of the I172A mutation on the kinetic parameters for the wild-type TIM-catalyzed reactions of the whole substrate and substrate pieces are consistent with a decrease in the basicity of the carboxylate side chain of Glu-167 for the mutant enzyme. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(C)) relative to an inactive open form (E(O)).

Dataset Information

The critical role of the loops of triosephosphate isomerase for its oligomerization, dynamics, and functionality.

Publications

The critical role of the loops of triosephosphate isomerase for its oligomerization, dynamics, and functionality.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets