Figure 1.
Chemical reaction catalyzed by MtUPRT.
Figure 2.
SDS-PAGE analysis of MtUPRT purification steps.
Lane 1, molecular weight protein marker; lane 2, crude extract; lane 3, sample loaded onto DEAE Sepharose CL6B column; lane 4, sample loaded onto Sephacryl S-200 column; lane 5, sample loaded onto Mono Q column; lane 6, homogeneous recombinant MtUPRT eluted from the Mono Q column.
Table 1.
Purification of MtUPRT from E. coli BL21(DE3) electrocompetent host cells.a
Figure 3.
Sedimentation equilibrium experiment.
A model (equation) of absorbance versus cell radius was fitted to the data by applying nonlinear regression. The experimental data for 1.5 mg/mL of protein at 9,000 and 11,000 rpm are shown. The random distribution of the residues (top panel) indicated a good quality fit in agreement with monomer-tetramer equilibrium.
Figure 4.
Calibration curve of Superdex 200 HR column with protein standards.
The following standards were employed (solid squares): ribonuclease A (13,700 Da), carbonic anhydrase (29,000 Da), ovalbumin (43,000 Da), conalbumin (75,000 Da), aldolase (158,000 Da), ferritin (440,000 Da) and thyroglobulin (669,000 Da). The Kav value was calculated for each standard protein using the equation (Ve – V0)/(Vt – V0), where is Ve the elution volume for the protein and Vt is the total bed volume, and Kav was plotted against the logarithm of standard molecular weights. The experimental Kav (open square) suggests a value of 109,650 Da for the molecular mass of recombinant MtUPRT in solution.
Figure 5.
Apparent steady-state kinetic parameters.
(A) Initial velocity of MtUPRT (U mg−1) as a function of increasing PRPP concentration in the presence of constant uracil concentration (10 µM). (B) Initial velocity of MtUPRT as a function of increasing uracil concentration in the presence of constant PRPP concentration (100 µM). (C) Initial velocity of MtUPRT as a function of increasing PRPP concentration in the presence of constant concentrations of uracil (10 µM) and GTP (100 µM). (D) Initial velocity of MtUPRT as a function of increasing uracil concentration in the presence of constant concentrations of PRPP (100 µM) and GTP (100 µM).
Figure 6.
Multiple sequence alignment of amino acid sequences of UPRTs from M. tuberculosis, B. caldolyticus, T. gondii, E. coli and S. solfataricus.
Amino acids for each polypeptide sequence were independently numbered. Identical conserved residues are indicated by stars below the alignment. Residues proposed to be involved in catalysis (ARg102 and Asp198), PRPP substrate binding (ARg77 and Arg102), and (or not) C-terminal glycine (Gly205) are highlighted (MtUPRT numbering). Multiple sequence alignment was carried out using Clustal W2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
Figure 7.
Evaluation of nucleotides as allosteric effectors.
All reactions contained 350 µM PRPP and 35 µM uracil. (•) standard reaction, (○) standard reaction containing 500 µM GTP, (□) standard reaction containing 500 µM CTP, (Δ) standard reaction containing 500 µM ATP, (×) standard reaction containing 500 µM UTP, (▪) standard reaction containing 100 µM UMP, (▴) standard reaction containing both 100 µM UMP and 500 µM CTP.
Figure 8.
Initial velocity patterns for MtUPRT.
Double-reciprocal plot of enzyme initial velocity−1 (mg U−1) versus [PRPP]−1 (µM−1). Concentrations of uracil were: 6 µM (open circles), 8 µM (filled triangle), and 10 µM (open squares).
Figure 9.
Isothermal titration (ITC) curves of binding of ligands to MtUPRT.
(A) Reverse titration of PRPP substrate. (B) Titration of uracil substrate. (C) Reverse titration of UMP product. (D) Titration of PPi product.
Table 2.
Thermodynamic parameters of PRPP and UMP ligands binding to MtUPRT.a
Figure 10.
Proposed kinetic mechanism for MtUPRT.
This order of substrate binding and product release is suggested on the basis of thermodynamic results.
Figure 11.
Dependence of kinetic parameters on pH.
(A) pH dependence of log kcat. (B) pH dependence of log kcat/KPRPP.