Abstract
Abstract In acidic environments, acid phosphatases (EC 3.1.3.2) play a crucial role in hydrolyzing phosphate ester linkages. Two distinct forms of acid phosphatase, designated AP-I and AP-II, were purified to homogeneity from Erythrina indica using a combination of gel filtration and affinity chromatographic techniques. The purification process involved multiple steps to ensure the enzymes were purified to homogenity, thereby facilitating detailed characterization. The active site of the purified AP-II was characterized in detail through chemical modification studies, which revealed the presence of one residue each of carboxylate, tryptophan, and serine. Substrate protection experiments using p-nitrophenyl phosphate effectively prevented the modification of all three residues, suggesting their essential role in the enzyme’s active site. These experiments provided strong evidence that these residues are directly involved in the catalytic process. Kinetic studies of the partially inactivated enzyme, achieved through the use of specific modifying agents Dicyclohexylcarbodiimide (DCHC) for carboxylate, N-Bromosuccinimide (NBS) for tryptophan, and Phenylmethylsulfonyl fluoride (PMSF) for serine, further confirmed the involvement of these residues in the catalytic mechanism. Additionally, statistical analysis of spectral data showed a good overall fit validating the experimental data. The results demonstrated that the inactivation of any of these residues significantly impaired the enzyme’s activity, highlighting their critical roles in the catalytic process. In addition, Circular dichroism (CD) analysis indicated similar secondary structural elements (α-helix and β-sheet) in AP-II. AP-II demonstrated a better polynomial fit (R² ≈ 0.89) indicating greater structural stability on performing data analysis using origin software. Furthermore, the three-dimensional structures of AP-II were predicted using computational (in silico) structural studies to investigate their structural organization. The results provide a comprehensive understanding of the active site architecture and the catalytic mechanism of AP-II, highlighting the critical roles of the carboxylate, tryptophan, and serine residues in the enzyme’s active function.