In homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM), cysteine 14 of each the two subunits forms part of the dimer interface. This residue is central for the catalysis and stability of TbTIM. Cys14 was changed to the other 19 amino acids to determine the characteristics that the residue must have to yield catalytically competent stable enzymes. C14A, C14S, C14P, C14T, and C14V TbTIMs were essentially wild type in activity and stability. Mutants with Asn, Arg, and Gly had low activities and stabilities. The other mutants had less than 1% of the activity of TbTIM. One of the latter enzymes (C14F) was purified to homogeneity. Size exclusion chromatography and equilibrium sedimentation studies showed that C14F TbTIM is a monomer, with a k(cat) approximately 1000 times lower and a K(m) approximately 6 times higher than those of TbTIM. In C14F TbTIM, the ratio of the elimination (methylglyoxal and phosphate formation) to isomerization reactions was higher than in TbTIM. Its secondary structure was very similar to that of TbTIM; however, the quantum yield of its aromatic residues was lower. The analysis of the data with the 19 mutants showed that to yield enzymes similar to the wild type, the residue must have low polarity and a van der Waals volume between 65 and 110 A(3). The results with C14F TbTIM illustrate that the secondary structure of TbTIM can be formed in the absence of intersubunit contacts, and that it has sufficient tertiary structure to support catalysis. Study holds ProTherm entries: 14876, 14877 Extra Details: 1 mM dithiothreitol and 1 mM azide were added in the experiments. dimer interface; activity; secondary structure; aromatic residues
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||3|
|Proteins||Triosephosphate isomerase, glycosomal ; Triosephosphate isomerase, glycosomal|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MSKPQPIAAANWKCNGSQQSLSELIDLFNSTSINHDVQCVVASTFVHLAMTKERLSHPKFVIAAQNAIAKSGAFTGEVSLPILKDFGVNWIVLGHSERRAYYGETNEIVADKVAAAVASGFMVIACIGETLQERESGRTAVVVLTQIAAIAKKLKKADWAKVVIAYEPVWAIGTGKVATPQQAQEAHALIRSWVSSKIGADVRGELRILYGGSVNGKNARTLYQQRDVNGFLVGGASLKPEFVDIIKATQ|