Cheleski Juliana, Freitas Renato F, Wiggers Helton José, Rocha Josmar R, de Araújo Ana Paula Ulian, Montanari Carlos A
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.
Protein Expr Purif. 2011 Apr;76(2):190-6. doi: 10.1016/j.pep.2010.11.013. Epub 2010 Dec 4.
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.
锥虫是有鞭毛的原生动物，可引发严重的寄生虫病，世界卫生组织已将这些疾病列为极为重要的热带病。一个备受关注的重要药物靶点是原生动物寄生虫克氏锥虫（恰加斯病的病原体）中的甘油醛-3-磷酸脱氢酶（克氏锥虫甘油醛-3-磷酸脱氢酶（TcGAPDH）；EC 1.2.1.12）。TcGAPDH是克氏锥虫糖酵解途径中的关键酶，催化D-甘油醛-3-磷酸（G3P）氧化磷酸化为1,3-二磷酸甘油酸（1,3-BPG），同时将氧化型烟酰胺腺嘌呤二核苷酸（NAD(+)）还原为还原型NADH。在此，我们描述了将克氏锥虫TcGAPDH基因克隆到pET-28a(+)载体中，在大肠杆菌中作为带标签蛋白进行表达、纯化及动力学表征。His(6)标签的TcGAPDH通过亲和层析进行纯化。采用分光光度法对重组His(6)-TcGAPDH进行酶活性测定以确定动力学参数。测定得到的D-甘油醛-3-磷酸和NAD(+)的表观米氏常数（K(M)(app)）分别为352±21和272±25 μM，这与文献中报道的无标签酶的值一致。我们通过等温滴定量热法（ITC）证明，这种载体修饰使酶活性在更长时间内得以保留。我们在此还报告了利用响应面法（RSM）来确定酶活性的最佳条件区域。RSM建立了一个二次模型，以pH和温度作为自变量来描述酶活性。根据RMS等高线图和方差分析，酶活性的最大值出现在29.1°C和pH 8.6时。高于37°C时酶活性开始下降这可能与先前报道的四级结构开始解体的过程有关。
