Variant Of Triosephosphate Isomerase Research Articles

Linear Free Energy Relationships for Enzymatic Reactions: Fresh Insight from a Venerable Probe.

ConspectusLinear free energy relationships (LFERs) for substituent effects on reactions that proceed through similar transition states provide insight into transition state structures. A classical approach to the analysis of LFERs showed that differences in the slopes of Brønsted correlations for addition of substituted alkyl alcohols to ring-substituted 1-phenylethyl carbocations and to the β-galactopyranosyl carbocation intermediate of reactions catalyzed by β-galactosidase provide evidence that the enzyme catalyst modifies the curvature of the energy surface at the saddle point for the transition state for nucleophile addition. We have worked to generalize the use of LFERs in the determination of enzyme mechanisms. The defining property of enzyme catalysts is their specificity for binding the transition state with a much higher affinity than the substrate. Triosephosphate isomerase (TIM), orotidine 5′-monophosphate decarboxylase (OMPDC), and glycerol 3-phosphate dehydrogenase (GPDH) show effective catalysis of reactions of phosphorylated substrates and strong phosphite dianion activation of reactions of phosphodianion truncated substrates, with rate constants kcat/Km (M–1 s–1) and kcat/KdKHPi (M–2 s–1), respectively. Good linear logarithmic correlations, with a slope of 1.1, between these kinetic parameters determined for reactions catalyzed by five or more variant forms of each catalyst are observed, where the protein substitutions are mainly at side chains which function to stabilize the cage complex between the enzyme and substrate. This shows that the enzyme-catalyzed reactions of a whole substrate and substrate pieces proceed through transition states of similar structures. It provides support for the proposal that the dianion binding energy of whole phosphodianion substrates and of phosphite dianion is used to drive the conversion of these protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes that show the same activity toward catalysis of the reactions of whole and phosphodianion truncated substrates. There is a good linear correlation, with a slope of 0.73, between values of the dissociation constants log Ki for release of the transition state analog phosphoglycolate (PGA) trianion and log kcat/Km for isomerization of GAP for wild-type and variants of TIM. This correlation shows that the substituted amino acid side chains act to stabilize the complex between TIM and the PGA trianion and that ca. 70% of this stabilization is observed at the transition state for substrate deprotonation. The correlation provides evidence that these side chains function to enhance the basicity of the E165 side chain of TIM, which deprotonates the bound carbon acid substrate. There is a good linear correlation, with a slope of 0.74, between the values of ΔG‡ and ΔG° determined by electron valence bond (EVB) calculations to model deprotonation of dihydroxyacetone phosphate (DHAP) in water and when bound to wild-type and variant forms of TIM to form the enediolate reaction intermediate. This correlation provides evidence that the stabilizing interactions of the transition state for TIM-catalyzed deprotonation of DHAP are optimized by placement of amino acid side chains in positions that provide for the maximum stabilization of the charged reaction intermediate, relative to the neutral substrate.

Phylogenetic spread of sequence data affects fitness of consensus enzymes: Insights from triosephosphate isomerase.

The concept of consensus in multiple sequence alignments (MSAs) has been used to design and engineer proteins previously with some success. However, consensus design implicitly assumes that all amino acid positions function independently, whereas in reality, the amino acids in a protein interact with each other and work cooperatively to produce the optimum structure required for its function. Correlation analysis is a tool that can capture the effect of such interactions. In a previously published study, we made consensus variants of the triosephosphate isomerase (TIM) protein using MSAs that included sequences form both prokaryotic and eukaryotic organisms. These variants were not completely native-like and were also surprisingly different from each other in terms of oligomeric state, structural dynamics, and activity. Extensive correlation analysis of the TIM database has revealed some clues about factors leading to the unusual behavior of the previously constructed consensus proteins. Among other things, we have found that the more ill-behaved consensus mutant had more broken correlations than the better-behaved consensus variant. Moreover, we report three correlation and phylogeny-based consensus variants of TIM. These variants were more native-like than the previous consensus mutants and considerably more stable than a wild-type TIM from a mesophilic organism. This study highlights the importance of choosing the appropriate diversity of MSA for consensus analysis and provides information that can be used to engineer stable enzymes.

Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase.

We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG⧧) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol–1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol–1, with a strong linear correlation between ΔG⧧ and ΔG° for all 11 variants, with slopes β = 0.73 (R2 = 0.994) and β = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.

Structural Analysis of an Epitope Candidate of Triosephosphate Isomerase in Opisthorchis viverrini

Opisthorchis viverrini, a parasitic trematode, was recategorized as a group 1 biological carcinogen because it causes opisthorchiasis, which may result in cholangiocarcinoma. A new strategy for controlling opisthorchiasis is needed because of issues such as drug resistance and reinfection. Triosephosphate isomerase (TIM), a key enzyme in energy metabolism, is regarded as a potential drug target and vaccine candidate against various pathogens. Here, we determined the crystal structures of wild-type and 3 variants of TIMs from O. viverrini (OvTIM) at high resolution. The unique tripeptide of parasite trematodes, the SAD motif, was located on the surface of OvTIM and contributed to forming a 310-helix of the following loop in a sequence-independent manner. Through thermal stability and structural analyses of OvTIM variants, we found that the SAD motif induced local structural alterations of the surface and was involved in the overall stability of OvTIM in a complementary manner with another parasite-specific residue, N115. Comparison of the surface characteristics between OvTIM and Homo sapiens TIM (HsTIM) and structure-based epitope prediction suggested that the SAD motif functions as an epitope.

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

Background: The glycolytic pathway plays an important role in tumor cells. Triosephosphate isomerase (TIM) catalyzes the reversible isomerization of D-glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) in the glycolysis. Proteomics of a human prostate adenocarcinoma cell line revealed the presence of the G233D TIM variant, a new allelic type whose biochemical properties have not been reported [1].Objective: Provide the first biochemical and biophysical characterization of the allelic variant G233D of TIM.Methods: The Michaelis-Menten curves using both substrates of TIM were obtained. Also the effect of the competitive inhibitor phosphoenolpyruvate (PEP) was assessed in presence of GAP and DHAP. The thermal stability in absence and presence of PEP was analyzed by circular dichroism spectroscopy. For comparison purposes, all the measurements were carried out on the wild type TIM and variant G233D.Results: The G233D variant exhibited a kcat value 4-fold lower than that of the WT enzyme in the GAP isomerization to DHAP, which is the reverse reaction of the glycolytic pathway. The G233D variant exhibited Ki and IC50 values of 120 μM and 356 μM in the presence of several concentrations of GAP and 0.3 mM DHAP, respectively. These inhibition parameters are similar to those exhibited by the WT enzyme. The thermal unfolding cooperativity of G233D variant was significantly increased upon PEP binding, suggesting that the ligand-bound enzyme was trapped in a rigid conformation.Conclusion: We suggest that the flow of GAP through glycolysis could be enhanced by the decreased activity of the G233D variant in the formation of DHAP.

Structure-based directed evolution of a monomeric triosephosphate isomerase: toward a pentose sugar isomerase.

Through structure-based and directed evolution approaches, a new catalytic activity has been established on the (β/α)8 barrel enzyme triosephosphate isomerase (TIM). This work started from ml8bTIM, a monomeric variant of TIM, in which the phosphate-binding loop (loop-8) had been shortened. Structure analysis suggested an additional point mutation (V233A), converting ml8bTIM into A-TIM. A-TIM has no detectable TIM activity, but it binds the TIM transition state analog, 2-phosphoglycollate. In an in vivo selection approach, we aimed at transferring the activity of three sugar isomerases (L-arabinose isomerase (L-AI), D-xylose isomerase A (D-XI) and D-ribose-5-phosphate isomerase (D-RPI)) onto A-TIM. Escherichia coli knockout variants were constructed, lacking E. coli L-AI, D-XI and D-RPI activities, respectively. Through a systematic approach, new A-TIM variants were obtained only from selection experiments with the L-AI knockout strain. Selection for D-RPI activity was impossible because of an impaired strain due to the gene knockouts. The selection for D-XI activity was unsuccessful, showing the importance of the starting protein for obtaining new biocatalytic properties. The L-AI-directed evolution experiments show that A-TIM already has residual in vivo L-AI activity. Most of the mutations providing A-TIM with enhanced L-AI activity are located in the loops between β-strands and the subsequent α-helices.

Stabilizing Proteins from Sequence Statistics: The Interplay of Conservation and Correlation in Triosephosphate Isomerase Stability

Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution. Consensus mutations have proven effective for increasing stability, but these mutations are successful only about half the time. We set out to understand why some consensus mutations fail to stabilize, and what criteria might be useful to predict stabilization more accurately. Overall, consensus mutations at more conserved positions were more likely to be stabilizing in our model, triosephosphate isomerase (TIM) from Saccharomyces cerevisiae. However, positions coupled to other sites were more likely not to stabilize upon mutation. Destabilizing mutations could be removed both by removing sites with high statistical correlations to other positions and by removing nearly invariant positions at which “hidden correlations” can occur. Application of these rules resulted in identification of stabilizing mutations in 9 out of 10 positions, and amalgamation of all predicted stabilizing positions resulted in the most stable yeast TIM variant we produced (+8 °C). In contrast, a multimutant with 14 mutations each found to stabilize TIM independently was destabilized by 2 °C. Our results are a practical extension to the consensus concept of protein stabilization, and they further suggest the importance of positional independence in the mechanism of consensus stabilization.

New allelic variant of triosephosphate isomerase found in cultured tumor cells of human prostate

A proteomic study of the proteins of the cell lines that model malignant and benign human prostate tumors (LNCaP, DU145, PC3, BPH-1) revealed a new allelic variant of the triosephosphate isomerase caused by the amino acid substitution 233G → D in the LNCaP. The study of the exon region 7 in TPI1 gene showed that the cells of the LNCaP line were heterozygous for a single-nucleotide substitution 2765 G/A, which gives rise to two gene expression products, such as allelic isoforms of triosephosphate isomerase that differ in the amino acid substitution 233G → D.

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove.

Triose Phosphate Isomerase Deficiency Is Caused by Altered Dimerization–Not Catalytic Inactivity–of the Mutant Enzymes

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative in vivo analyses of the enzymatic and functional properties of the different enzyme variants. We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations.

Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8) of triosephosphate isomerase: toward a new substrate specificity.

Loop 8 (residues 232-242) in triosephosphate isomerase (TIM) is a highly conserved loop that forms a tight binding pocket for the phosphate moiety of the substrate. Its sequence includes the fully conserved, solvent-exposed Leu238. The tight phosphate-binding pocket explains the high substrate specificity of TIM being limited to the in vivo substrates dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. Here we use the monomeric variant of trypanosomal TIM for exploring the structural consequences of shortening this loop. The mutagenesis, guided by extensive modeling calculations and followed up by crystallographic characterization, is aimed at widening the phosphate-binding pocket and, consequently, changing the substrate specificity. Two new variants were characterized. The crystal structures of these variants indicate that in monomeric forms of TIM, the Leu238 side-chain is nicely buried in a hydrophobic cluster. Monomeric forms of wild-type dimeric TIM are known to exist transiently as folding intermediates; our structural analysis suggests that in this monomeric form, Leu238 of loop 8 also adopts this completely buried conformation, which explains its full conservation across the evolution. The much wider phosphate-binding pocket of the new variant allows for the development of a new TIM variant with a different substrate specificity.