Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry.
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.
Cheleski
Juliana
J
Grupo de Química Medicinal de Produtos Naturais, NEQUIMED-PN, Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil.
Wiggers
Helton José
HJ
Citadini
Ana Paula
AP
da Costa Filho
Antônio José
AJ
Nonato
Maria Cristina
MC
Montanari
Carlos Alberto
CA
D055162:Biocatalysis; D002151:Calorimetry / Q000379:methods*; D000090722:Dihydroorotate Dehydrogenase; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D057075:Enzyme Assays; D005650:Fumarates / Q000378:metabolism; D007700:Kinetics; D017830:Octoxynol / Q000737:chemistry; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014349:Trypanosoma cruzi / Q000201:enzymology*
D000090722:Dihydroorotate Dehydrogenase; D005650:Fumarates; D011994:Recombinant Proteins; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D017830:Octoxynol; D044925:Oxidoreductases Acting on CH-CH Group Donors; D004121:Dimethyl Sulfoxide