Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme

Biochemistry
Gloria Hernández-AlcántaraR Pérez-Montfort

Abstract

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 ...Continue Reading

References

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Citations

Jul 20, 2002·Biochemical and Biophysical Research Communications·Alfredo Téllez-ValenciaArmando Gómez-Puyou
Dec 14, 2007·Molecular and Biochemical Parasitology·Sergio Enriquez-FloresHoracio Reyes-Vivas
May 18, 2004·Proteins·Gabriel López-VelázquezHoracio Reyes-Vivas
Jan 16, 2007·Proteins·Viviana Zomosa-SignoretArmando Gómez-Puyou
Nov 11, 2006·Journal of Molecular Biology·Horacio Reyes-VivasGabriel Lopez-Velazquez
Oct 1, 2018·Biochemical and Biophysical Research Communications·Timothy C ChangJason P Schwans

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