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Magnitude and origin of the enhanced basicity of the catalytic glutamate of triosephosphate isomerase.

ABSTRACT: Glu-167 of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM) acts as the base to deprotonate substrate to form an enediolate phosphate trianion intermediate. We report that there is a large ~6 pK unit increase in the basicity of the carboxylate side chain of Glu-167 upon binding of the inhibitor phosphoglycolate trianion (I(3-)), an analog of the enediolate phosphate intermediate, from pKEH ? 4 for the protonated free enzyme EH to pK(EHI) ? 10 for the protonated enzyme-inhibitor complex EH•I(3-). We propose that there is a similar increase in the basicity of this side chain when the physiological substrates are deprotonated by TbbTIM to form an enediolate phosphate trianion intermediate and that it makes an important contribution to the enzymatic rate acceleration. The affinity of wildtype TbbTIM for I(3-) increases 20,000-fold upon decreasing the pH from 9.3 to 4.9, because TbbTIM exists mainly in the basic form E over this pH range, while the inhibitor binds specifically to the rare protonated enzyme EH. This reflects the large increase in the basicity of the carboxylate side chain of Glu-167 upon binding of I(3-) to EH to give EH•I(3-). The I172A mutation at TbbTIM results in an ~100-fold decrease in the affinity of TbbTIM for I(3-) at pH < 6 and an ~2 pK unit decrease in the basicity of the carboxylate side chain of Glu-167 at the EH•I(3-) complex, to pK(EHI) = 7.7. Therefore, the hydrophobic side chain of Ile-172 plays a critical role in effecting the large increase in the basicity of the catalytic base upon the binding of substrate and/or inhibitors.

SUBMITTER: Malabanan MM

PROVIDER: S-EPMC3649849 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Magnitude and origin of the enhanced basicity of the catalytic glutamate of triosephosphate isomerase.

Malabanan M Merced MM Nitsch-Velasquez Lucia L Amyes Tina L TL Richard John P JP

Journal of the American Chemical Society 20130410 16

Glu-167 of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM) acts as the base to deprotonate substrate to form an enediolate phosphate trianion intermediate. We report that there is a large ~6 pK unit increase in the basicity of the carboxylate side chain of Glu-167 upon binding of the inhibitor phosphoglycolate trianion (I(3-)), an analog of the enediolate phosphate intermediate, from pKEH ≈ 4 for the protonated free enzyme EH to pK(EHI) ≈ 10 for the protonated enzyme-inhibitor ...[more]

PMID: 23560625

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Project description:Kinetic parameters are reported for the reactions of whole substrates (kcat/Km, M(-1) s(-1)) (R)-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) and for the substrate pieces [(kcat/Km)E·HPi/Kd, M(-2) s(-1)] glycolaldehyde (GA) and phosphite dianion (HPi) catalyzed by the I172A/L232A mutant of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM). A comparison with the corresponding parameters for wild-type, I172A, and L232A TbbTIM-catalyzed reactions shows that the effect of I172A and L232A mutations on ?G(?) for the wild-type TbbTIM-catalyzed reactions of the substrate pieces is nearly the same as the effect of the same mutations on TbbTIM previously mutated at the second side chain. This provides strong evidence that mutation of the first hydrophobic side chain does not affect the functioning of the second side chain in catalysis of the reactions of the substrate pieces. By contrast, the effects of I172A and L232A mutations on ?G(?) for wild-type TbbTIM-catalyzed reactions of the whole substrate are different from the effect of the same mutations on TbbTIM previously mutated at the second side chain. This is due to the change in the rate-determining step that determines the barrier to the isomerization reaction. X-ray crystal structures are reported for I172A, L232A, and I172A/L232A TIMs and for the complexes of these mutants to the intermediate analogue phosphoglycolate (PGA). The structures of the PGA complexes with wild-type and mutant enzymes are nearly superimposable, except that the space opened by replacement of the hydrophobic side chain is occupied by a water molecule that lies ?3.5 Å from the basic side chain of Glu167. The new water at I172A mutant TbbTIM provides a simple rationalization for the increase in the activation barrier ?G(?) observed for mutant enzyme-catalyzed reactions of the whole substrate and substrate pieces. By contrast, the new water at the L232A mutant does not predict the decrease in ?G(?) observed for the mutant enzyme-catalyzed reactions of the substrate piece GA.

Project description:BackgroundWe have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.ResultsIn this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.ConclusionsThe results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.

Dataset Information

Magnitude and origin of the enhanced basicity of the catalytic glutamate of triosephosphate isomerase.

Publications

Magnitude and origin of the enhanced basicity of the catalytic glutamate of triosephosphate isomerase.

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