acid-phosphatase and phosphohistidine

After reporting the mechanisms of purple acid phosphatases against acid environments and alkaline phosphatases against alkaline environments, in the present work, we continued investigating the relationship between catalytic structures of histidine acid phosphatases (HAPs) and acid environments. On the basis of the comparison of the crystal structures of several HAP members, a series of models were constructed and calculated using density functional theory. Our calculations describe a complete catalytic cycle of HAPs, including a free stage and a catalytic reaction stage. This cycle reveals a definite mechanism for HAPs to survive in acidic environments, which can be used to nicely interpret acidic pH optima of HAPs. It also suggests that a free water molecule from a solvent should be the nucleophile for hydrolyzing the phosphohistidine intermediate. Our studies are focused on the biological significance of enzymatic mechanisms and raise two concrete criteria: the logic-complete catalytic cycle and the evolutional relation with family members and molecular environments.

Topics: Acid Phosphatase; Biocatalysis; Catalytic Domain; Density Functional Theory; Histidine; Hydrolysis; Kinetics; Models, Molecular

Calculation of the free energy profile for hydrolysis of phosphohistidine using ONIOM methodology indicates a much tighter transition state in the enzyme active site compared to that in explicit water and elucidates the role of active site residues in catalysis.

Topics: Acid Phosphatase; Biocatalysis; Catalytic Domain; Histidine; Hydrolysis; Protein Tyrosine Phosphatases; Water

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 carbethoxylation of prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was accompanied by modification of histidine residues and the inactivation of the enzyme. These findings are consistent with photoinactivation experiments described earlier (Rybarska, J. and Ostrowski, W (1974) Acta Biochim, Polon. 21, 377--390). Prostatic acid phosphatase was phosphorylated at alkaline pH using p-nitrophenyl [32P]phosphate as substrate. Phosphoryl enzyme is stable in alkaline solutions and undergoes dephosphorylation at acidic pH. After hydrolysis of phosphoryl enzyme in strong alkaline solution, a single phosphoryl amino acid was isolated from hydrolyzate and identified as the tau-phosphohistidine.

Topics: Acid Phosphatase; Drug Stability; Histidine; Humans; Hydrogen-Ion Concentration; Male; Phosphorylation; Prostate; Prostatic Hyperplasia