RESUMEN
The purple acid phosphatase was purified from 5.9-fold to apparent homogeneity from Anagelis arvensis seeds using SP-Sephadex C-50 and Sephadex G-100 chromatography. The results of residual activity tests conducted using different temperature ranges (50-70 °C) were calculated as the activation energy (Ed = 72 kJ/mol), enthalpy (69.31 ≤ (ΔH° ≤ 69.10 kJ/mol), entropy (-122.48 ≤ ΔS° ≤ -121.13 J/mol·K), and Gibbs free energy (108.87 ≤ ΔG° ≤ 111.25 kJ/mol) of the enzyme irreversible denaturation. These thermodynamic parameters indicate that this novel PAP is highly thermostable and may be significant for use in industrial applications. However, it may be confirmed by stopped-flow measurements that this substitution produces a chromophoric Fe3+ site and a Pi-substrate interaction that is about ten times faster. Additionally, these data show that phenyl phosphate hydrolysis proceeds more rapidly in metal form of A. arvensis PAP than the creation of a µ-1,3 phosphate complex. The Fe3+ site in the native Fe3+-Mn2+ derivative interacts with it at a faster rate than in the Fe3+-Fe2+ form. This is most likely caused by a network of hydrogen bonds between the first and second coordination spheres. This suggests that the choice of metal ions plays a significant role in regulating the activity of this enzyme.
RESUMEN
The AcPase exhibits a specific activity of 31.32 U/mg of protein with a 728-fold purification, and the yield of the enzyme is raised to 3.15 %. The Zn2+-dependent AcPase showed a purification factor of 1.34 specific activity of 14 U/mg of proteins and a total recovery of 5.14. The SDS-PAGE showed a single band corresponding to a molecular weight of 18 kDa of AcPase and 29 kDa of Zn2+-dependent AcPase. The AcPase enzyme has shown a wide range of substrate specificity for p-NPP, phenyl phosphate and FMN, while in the case of ZnAcPase α and ß-Naphthyl phosphate and p-NPP were proved to be superior substrates. The divalent metal ions like Mg2+, Mn2+, and Ca2+ increased the activity, while other substrates decreased the enzyme activity. The Km (0.14 mM) and Vmax (21 µmol/min/mg) values of AcPase were higher than those of Zn2+-AcPase (Km = 0.5 mM; Vmax = 9.7 µmol/min/mg). The Zn2+ ions activate the Zn2+-AcPase while Fe3+, Al3+, Pb2+, and Hg2+ showed inhibition on enzyme activity. Molybdate, vanadate and phosphate were found to be competitive inhibitors of AcPase with Ki values 316 µM, 185 µM, and 1.6 mM, while in Zn2+-AcPase tartrate and phosphate also showed competitive inhibition with Ki values 3 mM and 0.5 mM respectively.