The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45degreesC using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55degreesC. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60degreesC. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60degreesC, with the reaction products inorganic phosphate - P (10 mM) and p-nitrophenol - p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (T-m) value of 75degreesC and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.26641671111

Purification and kinetic proper ties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean (Ricinus communis L., IAC-80) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2000-fold to homogeneity, with a final specific activity of 3.8 mu kat mg(-1) protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa, The acid phosphatase had a pH optimum of 5.5 and an apparent K-m value for p-nitrophenylphosphate of 0.52 mM, The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p-chloromercuribenzoate (pCMB), Cu2+ and Zn2+, The strong inhibition by pCMB, Cu2+ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (PPi) as substrate. The highest specificity constant (V-max/K-m) was observed with PPi, making it a potential physiological substrate.107215115

Essential sulfhydryl groups in the active site of castor bean (Ricinus communis) seed acid phosphatase

In order to determine which amino acids are involved in substrate binding, castor bean seeds acid phosphatase was treated with amino acid-modifying reagents, such as phenylglyoxal (PGO), iodoacetic acid (IAA), N-bromosuccinimide (NBS), N-acetylimidazole (NAI), diethylpyrocarbonate (DEPC), N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), specific for arginine, cysteine, tryptophan, tyrosine, histidine, and aspartic and glutamic acids, respectively. Enzyme activity was determined using p-nitrophenylphosphate (pNPP) as substrate. Enzyme inhibition was observed with IAA, NBS, EDC and DCCD. In the presence of the reaction products, p-nitrophenol (pNP) and inorganic phosphate (Pi), which are competitive inhibitors, the enzyme was protected from inactivation by IAA, indicating involvement of the enzyme active site. The inhibition by IAA was time- and concentration-dependent, with an apparent bimolecular rate constant of 48 x 10(-4) M-1 s(-1),and two molecules of IAA bound per active site. Our results suggest that sulfhydryl groups are essential for enzyme catalysis, and are located in or near the substrate-binding domain. Other amino acids such as tryptophan, aspartic and glutamic acids, were also important for the enzyme activity, but were probably located outside of the active site. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.164462963

Inhibition of bovine kidney low molecular mass phosphotyrosine protein phosphatase by uric acid

Uric acid inhibited 50% of the activity of bovine kidney low molecular mass phosphotyrosine protein phosphatase at concentrations of 1.0, 0.4, 1.3, and 0.2 mM, respectively for p -nitrophenyl phosphate (p -NPP), flavine mononucleotide, beta-naphthyl phosphate and tyrosine phosphate (Tyr-P) as substrates. The mixed type inhibition of p -NPP hydrolysis was fully reversible, with K-ic and K-iu values of 0.4 and 1.1 mM, respectively; the inhibition by uric acid shifted the pH optimum from 5.0 to 6.5. When Tyr-P was the substrate, competitive inhibition was observed with a K-i value of 0.05 mM. Inhibition studies by uric acid in the presence of thiol compounds, and preincubation studies in the presence of inorganic phosphate suggest that the interaction of uric acid with the enzyme occurred at the active site, but did not involve SH residues, and that the mechanism of inhibition depended on the structure of the substrates.17534535

Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase

In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaG(u), was calculated at 25 degreesC as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole. (C) 2004 Elsevier Ltd. All rights reserved.65783183

Electroanalytical determination of acid phosphatase activity by monitoring p-nitrophenol

The development of a modified electrode for the selective determination of p-nitrophenol (PNP) to be applied in the determination of the enzymatic activity (EA) of acid phosphatase using p-nitrophenylphosphate (PNPP) as substrate is described. Elimination of the interference of PNPP was made by covering the electrode surface using 25 mul of a 5% (m/v) Nafion (R) solution. In comparison to the glassy carbon (GC) electrode without modification, the presence of the membrane promoted a reduction in the sensitivity and an increase in the detection limit for the determination of PNP, in addition to a decrease of the electron transfer constant. The GC electrode covered with a Nafion (R) membrane showed a linear response range between 20 and 230 mu mol l(-1) for PNP, adjusted by the equation I-p = 0.157 + 0.0089 [PNP], for N = 22 with a correlation coefficient of 0.9984. This electrode was used for the determination of the EA of acid phosphatase from castor bean seeds and an activity of 38.40 U mg(-1) was found. (C) 2001 Elsevier Science B.V. All rights reserved.441220721