Connecting Active‐Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme

Abstract

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms.

Leptospira interrogans triosephosphate isomerase: exploring the structural determinants of stability, high reaction rate and specificity.

“…We also determined the kinetic constants for two of the extensively studied pathogenic mesophilic TIMs viz., Trypanosoma brucei TIM, TbTIM, (k cat = 945 s −1 ; K m = 0.17 mM) and Plasmodium falciparum TIM, PfTIM, (k cat = 500 s −1 ; K m = 0.9 mM) under the same assay conditions at 25 °C (Table 1). The PfTIM and TbTIM kinetic parameters were in agreement with previous studies [33,52]. Pairwise sequence alignment (ClustalOmega online server [53]) of the three protein sequences (SwissProt online database [54]) showed that the sequence identity between LiTIM-TbTIM is 35%, LiTIM-PfTIM is 40% and PfTIM-TbTIM is 42% out of which around 28% residues are identical between all the three, ( Figure S2).…”

The role of ligand-gated conformational changes in enzyme catalysis

“…Finally, the importance of loop 6 flexibility to efficient catalysis can also be seen in mutational studies of rare variations of position 96 (proline in the wild-type) of the TIM from Plasmodium falciparum ( Pf TIM) [73], which is analogous to the proline at position 168 in Tbb TIM [34]. In both cases, it is clear that this residue plays an important role in modulating the conformational transition of loop 6, and, most critically, in positioning E165 ( y TIM numbering) in a catalytically competent position in the TIM active site (Figure 2).…”

A guide to the effects of a large portion of the residues of triosephosphate isomerase on catalysis, stability, druggability, and human disease

“…studied F96 ( 95 ) in Plasmodium falciparum PfTIM. Using crystallographic data of different mutants in this position (S, H, Y, A and W) they concluded that a change in the water network in the catalytic site is affecting the properties of the enzyme and that residue 96 is optimizing the position of the catalytic residue E165 ( 164 ) . The inhibiting effect of some dyes on the activity of PfTIM was tested; a direct interaction of some of them with F96 was shown, suggesting that the quite unique F96 ( 95 ) of PfTIM could be a good target for drug design .…”