Probing the role of highly conserved residues in triosephosphate isomerase--analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme.
UNLABELLED
Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodium falciparum triosephosphate isomerase (PfTIM) enzyme (EC 5.3.1.1) as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of Kd values for dimer dissociation (Q64N = 73.7 ± 9.2 nm and Q64E = 44.6 ± 8.4 nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 °C and 45 °C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity.


DATABASE
Structural data have been deposited in the Protein Data Bank under accession numbers 4ZZ9, 5BMW, 5BMX, 5BNK and 5BRB.
['Bandyopadhyay|Debarati|D|', 'Murthy|Mathur R N|MR|', 'Balaram|Hemalatha|H|', 'Balaram|Padmanabhan|P|']
D000595:Amino Acid Sequence D019943:Amino Acid Substitution D055162:Biocatalysis D017124:Conserved Sequence D030562:Databases, Protein D019281:Dimerization D004795:Enzyme Stability D018698:Glutamic Acid D005973:Glutamine D006358:Hot Temperature D006860:Hydrogen Bonding D006863:Hydrogen-Ion Concentration D008958:Models, Molecular D016297:Mutagenesis, Site-Directed D050505:Mutant Proteins D010963:Plasmodium falciparum D011487:Protein Conformation D015800:Protozoan Proteins D011994:Recombinant Proteins D013912:Threonine D014305:Triose-Phosphate Isomerase
conserved residues in enzymes triosephosphate isomerase amino acid conservation enzyme structure and function mutational effects on triosephosphate isomerase