Structure and kinetics of the enzyme glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi and mutants D210L, D2101-G213D / Estudos estruturais e cinéticos da enzima gliceraldeido-3-fosfato desidrogenase de trypanosoma cruzi e mutantes D21OL,D21OL-G213D

AUTOR(ES)

Beatriz Gomes Guimarães

DATA DE PUBLICAÇÃO

1998

RESUMO

The glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Typanosoma cruzi and its mutants D210L and D210L-G213D were expressed in E. coli and purified, followed by kinetic and crystallization assays. Both wild type enzyme and D210L-G213D mutant were crystallized in P21 space group and the crystals presented good X-ray diffraction patterns. The three-dimensional structure of the wild type enzyme was determined at 2.5 and 2.15 A resolution from data collected at 277 and 100 K respectively. The structures were refined to a crystallographic R-factor of 16.0% for data collected at 277 K and 18.8% for those collected at 100 K. Also, the structure of the D210L-G213D mutant was solved at 2.15 A resolution and refined to a crystallographic R-factor of 18.9% with good geometry indicators. Comparison between the wild type enzyme structures solved at both temperatures led to interesting results concerning the crystal packing. Flash-cooled crystals presented a 10.5% shrink in the unit cell volume being the major reduction observed in the parameter a (14.5%). Superposition of the unit cells showed a global rotation of the asyrnmetric unit content of about 5 degrees around an axis approximately parallel to b. On the other hand, the analysis of the wild type and mutant enzyme structures, together with the kinetic parameters, allowed a discussion about some catalytic mecanism details, mainly the role of the Arg249 residue. The results showed that this residue might be involved in the reorientation of one of the intermediates during the catalytic process.

ASSUNTO(S)

cinética enzimática knetic chagas disease t.cruzi t. cruzi crystal structure doença de chagas gapdh estrutura cristalográfica gapdh

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