Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.
Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 μM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly.
Grupo de Estudos em Química Medicinal de Produtos Naturais-NEQUIMED-PN, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Juliana
J
Cheleski
Renato F
RF
Freitas
Helton José
HJ
Wiggers
Josmar R
JR
Rocha
Ana Paula Ulian
AP
de Araújo
Carlos A
CA
Montanari
D002151:Calorimetry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D005986:Glyceraldehyde 3-Phosphate; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)