Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry
| Autor: | Antonio José da Costa Filho, Maria Cristina Nonato, Ana Paula da Silva Citadini, Carlos A. Montanari, H.J. Wiggers, Juliana Cheleski |
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| Rok vydání: | 2009 |
| Předmět: |
Oxidoreductases Acting on CH-CH Group Donors
Stereochemistry Octoxynol Trypanosoma cruzi Biophysics Dihydroorotate Dehydrogenase Calorimetry Biochemistry Michaelis–Menten kinetics Substrate Specificity symbols.namesake Fumarates CINÉTICA QUÍMICA Dimethyl Sulfoxide Enzyme kinetics Molecular Biology Enzyme Assays chemistry.chemical_classification Orotic Acid biology Chemistry Active site Isothermal titration calorimetry Cell Biology Arrhenius plot Recombinant Proteins Gibbs free energy Kinetics Enzyme biology.protein symbols Dihydroorotate dehydrogenase Biocatalysis Thermodynamics |
| Zdroj: | Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 1096-0309 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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