A comparative study on active site analysis of the two forms of acid phosphatases AP-I and AP-II purified from the seeds of the medicinally important plant Erythrina indica
摘要
Acid phosphatase (EC 3.1.3.2) is an enzyme that catalyzes the cleavage of phosphate ester linkages under acidic conditions. This research describes the isolation and purification of two isozymes, AP-I and AP-II, from Erythrina indica seeds using gel filtration and affinity chromatography techniques. The characterization of the active sites of the purified AP-I and AP-II isozymes was carried out using chemical modification experiments, which indicated the existence of one carboxylate, tryptophan, and serine residue in both AP-I and AP-II isozymes. The substrate protection experiments involving p-nitrophenyl phosphate resulted in the protection of all three residues against modification by Dicyclohexylcarbodiimide (carboxylate), N-bromosuccinimide (tryptophan), and phenylmethylsulfonyl fluoride (serine). The kinetic analysis of partially inactivated enzymes through modification by these agents showed their involvement in catalysis. The effect of the environment surrounding the tryptophan residues in the active sites of both isozymes was investigated using fluorescence spectroscopy technique. The obtained data was analyzed using Stern-Volmer equation to determine the values of Ksv and Kq. It was observed that the fraction of tryptophan residues accessible was one using modified Stern-Volmer equation. From the experiment results, it can be observed that the microenvironment around tryptophan amino acids is negative charged, since the neutral quencher acrylamide and positive quencher cesium chloride showed higher quenching effect compared to positive quencher potassium iodide. CD analysis suggests the tertiary structure is made up of 45% alpha helical, 23% beta pleated, and 32% random coils.