acid-phosphatase and phenylphosphate

Histidine phosphatases are a class of enzymes that are characterized by the presence of a conserved RHGXRXP motif. This motif contains a catalytic histidine that is being phosphorylated in the course of a dephosphorylation reaction catalyzed by these enzymes. Prostatic acid phosphatase (PAP) is one such enzyme. The dephosphorylation of phosphotyrosine by PAP is a two-step process. The first step involves the transfer of a phosphate group from the substrate to the histidine (His12). The present study reports on the details of the first step of this reaction, which was investigated using a series of quantum chemistry calculations. A number of quantum models were constructed containing various residues that were thought to play a role in the mechanism. In all these models, the transition state displayed an associative character. The transition state is stabilized by three active site arginines (Arg11, Arg15, and Arg79), two of which belong to the aforementioned conserved motif. The work also demonstrated that His12 could act as a nucleophile. The enzyme is further characterized by a His257-Asp258 motif. The role of Asp258 has been elusive. In this work, we propose that Asp258 acts as a proton donor which becomes protonated when the substrate enters the binding pocket. Evidence is also obtained that the transfer of a proton from Asp258 to the leaving group is possibly mediated by a water molecule in the active site. The work also underlines the importance of His257 in lowering the energy barrier for the nucleophilic attack.

Topics: Acid Phosphatase; Binding Sites; Computer Simulation; Guanidines; Histidine; Imidazoles; Models, Molecular; Organophosphates; Phosphorylation; Protein Tyrosine Phosphatases; Quantum Theory; Water

The steady-state kinetics of hydrolysis reaction catalysed by human prostatic acid phosphatase (PAP) by using 1-naphthyl phosphate, phenyl phosphate and phosphotyrosine as substrates has been studied at pH 5.5. The substrate binding curves were sigmoidal and Hill cooperation coefficient h was higher than 1 for each of the examined compounds. Thus, human prostatic acid phosphatase kinetics exhibits positive cooperativity towards the studied substrates. The extent of cooperativity was found to depend on the substrate used and on enzyme concentration. The highest cooperativity of PAP was observed for 1-naphthyl phosphate and the lowest for phosphotyrosine. When prostatic phosphatase concentration increased, Hill cooperation coefficient (h) and half saturation constant (K(0.5)) both grew, but the catalytic constant (k(cat)) remained constant, for each of the substrates studied. Ligand-induced association-dissociation equilibrium of the active oligomeric species (monomer-dimer-tetramer-oligomers) is suggested.

Topics: Acid Phosphatase; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Male; Models, Theoretical; Naphthalenes; Organophosphates; Organophosphorus Compounds; Phosphotyrosine; Prostate

A continuous spectrophotometric assay for the determination of the initial rate of an acid phosphatase-catalyzed reaction in an acidic environment, using phenyl phosphate as a substrate, is presented. The method is based on the continuous determination of phenol, a product of the enzymatic hydrolysis, by the kinetic measurement of its absorbance. The method allows for the direct estimation of acid phosphatase activity in an acidic solution. This is possible without interrupting the reaction by alkalization or precipitation required for commonly used end-point colorimetric detection procedures for phosphate or phenol, and without the use of any coupled assays. The method has been developed for acid phosphatase activity determination in an aqueous solution and in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles in a broad pH range (pH 3.8 to 8.8). The proposed procedure has been used for the determination of kinetic constants (K(m) and kcat) for human prostatic acid phosphatase in aqueous solutions, and in AOT-isooctane-water reverse micelles, at pH 3.8, 4.5, and 5.7.

Topics: Acid Phosphatase; Dioctyl Sulfosuccinic Acid; Humans; Hydrogen-Ion Concentration; Kinetics; Male; Micelles; Octanes; Organophosphates; Prostate; Spectrophotometry, Ultraviolet; Water

1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity. 2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5-20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein. 3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein. 4. The rat testis AcPase IV is a metal activated enzyme in which Mg2+ is the metal activating agent with a Ka = 0.88 x 10(-3) M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg2+ ions, is 0.23 x 10(-3) M. 5. The enzyme preferentially hydrolyzes p-nitrophenylphosphate, phenylphosphate and ATP.

Topics: Acid Phosphatase; Adenosine Triphosphate; Animals; Binding Sites; Cations, Divalent; Centrifugation, Density Gradient; Chromatography, Gel; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Magnesium; Male; Molecular Weight; Nitrophenols; Organophosphates; Organophosphorus Compounds; Rats; Sepharose; Testis

4-Difluoromethylphenyl bis(cyclohexylammonium) phosphate was synthesized in 4 steps starting from dibenzyl phosphite and shown to be a time-dependent suicide inactivator of human prostatic acid phosphatase and the SHP protein tyrosine phosphatase. The inactivation of human prostatic acid phosphatase followed pseudo-first-order kinetics with inactivation constants of Ki = 1.0 mM; ki = 0.15 min-1 (t1/2 = 4.6 min at saturation). Phenyl phosphate protected the enzyme against inactivation, indicating that inactivation occurs in the active site. The inactivation of SHP also followed pseudo-first-order kinetics, with a t1/2 = approximately 15 min in the presence of 8.2 mM inhibitor. The mechanism of inactivation likely involves the enzymatic release of difluoromethyl phenol which rapidly eliminates fluoride, generating a quinone methide. This potent electrophile then reacts with residues at the active site of the enzyme. This inhibitor and peptidic derivatives thereof have excellent potential for selective inactivation and labeling of protein tyrosine phosphatases.

Topics: Acid Phosphatase; Humans; Indicators and Reagents; Kinetics; Male; Molecular Structure; Organophosphates; Prostate; Protein Tyrosine Phosphatases; Recombinant Proteins

Topics: Acid Phosphatase; Animals; Biochemical Phenomena; Humans; Hydrolysis; Inferior Colliculi; Male; Organophosphates; Phosphates; Phosphoric Monoester Hydrolases; Rabbits; Testis