Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry.
Anal Biochem
; 399(1): 13-22, 2010 Apr 01.
Article
em En
| MEDLINE
| ID: mdl-19932077
Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K(M)) and maximum velocity (V(max)) for both of the TcDHODH substrates: dihydroorotate (K(M)=8.6+/-2.6 microM and V(max)=4.1+/-0.7 microMs(-1)) and fumarate (K(M)=120+/-9 microM and V(max)=6.71+/-0.15 microMs(-1)). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K(M). Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Trypanosoma cruzi
/
Calorimetria
/
Oxirredutases atuantes sobre Doadores de Grupo CH-CH
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Estados Unidos