Probing the Role of Highly Conserved Residues in Triosephosphate Isomerase : Biochemical & Structural Investigations

Conserved residues in protein are crucial for maintaining structure and function, either by direct involvement in chemistry or indirectly, by being essential for folding, stability and oligomerisation and are mostly clustered near active sites. The variability of sequences of the same protein from diverse organisms is a reflection of the selective pressures of evolution. Sequence conservation analysis with 3397 bacterial triosephosphate isomerase (TIM) sequences using Plasmodium falciparum (Pf) TIM as template, showed full conservation of ten residues, K12, T75, H95, E97, C126, E165, P166, G209, G210 and G228. The integrity of the enzyme active site, which lies near the dimer interface, makes TIM an obligatory dimer. Attempts to engineer active monomeric TIM have not been successful. The present study assesses the effects of mutations at fully conserved position 75 (Thr) and the highly conserved position 64 (Q: 3011, E: 383) near the dimer interface, using the recombinant Plasmodial enzyme. Residue 64, Gln in Pf, and T75 interact with the catalytic E97 and K12, respectively. Preliminary analysis of available crystal structures showed that Gln 64 takes part in a single intersubunit interaction and maintains the obligatory strained backbone angles of the catalytic K12 residue, while Thr 75 is involved in four intersusunit hydrogen bond interactions. This led to the hypothesis that mostly, Gln at position 64 is crucial for enzyme activity and Thr at position 75 for the integrity of the dimer. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The major findings revealed that the mutations at position 64 have a significant effect on dimer integrity with a 1000 fold increase in the dimer dissociation constant compared to the wild type enzyme, while dimer stability was unimpaired for the T75 mutants. Concentration dependence of activity yielded an estimate of dimer dissociation constant (Kd) values (Q64N 73.7±9.2 nM and Q64E 44.6±8.4 nM). Enzyme activity values of the T75 mutants are comparable to the wild type, except for T75N which shows a 4-fold drop in activity. All four T75 mutants show a dramatic fall in activity between 35 °-45 °C. Crystal structure determination of the T75S/V/N mutant offers insights into the variation in local interactions with T75N showing the largest changes. These results were unanticipated emphasising the uncertainties involved in inferring functional and structural role for individual residues based only on analysis of interactions observed in crystal structures. Nanospray ionisation mass spectrometric studies has also been used to probe the oligomeric properties of the three mutant proteins Q64N, Q64E and T75S and the wild type enzyme in the gas phase. The gas phase distributions of dimeric and monomeric species have been examined under a wild range of collision energies (40 – 160 eV). The order in the gas phase, PfTIM wild type > T75S > Q64E ~ Q64N, together with the solution phase experiments described above establish the importance of Q64 and T75 in influencing stability and activity. Inhibition studies with a 27 residue synthetic dimer interface peptide and the Q64 mutants establish that the interaction between the protein and the peptide was facilitated in the case of monomeric species