Hydrolysis Of Phosphate Monoesters Research Articles

Kinetic and Mechanistic Characterization of a Mammalian Protein-tyrosine Phosphatase, PTP1

The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat protein-tyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques. Product inhibition and 18O exchange experiments are consistent with the enzymatic reaction proceeding through two chemical steps, i.e. formation and breakdown of a covalent phosphoenzyme intermediate. The variation of kcat/Km with pH indicates that three ionizable groups are involved in enzyme substrate binding and catalysis. The first group must be deprotonated and is attributed to the second ionization of the substrate. The other two groups with pK alpha values of 5.1 and 5.5 correspond to two enzyme active site residues. The kcat-pH profiles for both p-nitrophenyl phosphate and beta-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK alpha values derived from the acidic limb and the basic limb of the profile being 4.4 and 6.8, respectively. This suggests that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate at all pH values. Results from leaving group dependence of kcat at two different pH values support the above conclusion. Furthermore, burst kinetics have been demonstrated with PTP1 using p-nitrophenyl phosphate as a substrate. The rate constants for the formation and the breakdown of the intermediate are 241 and 12 s-1, respectively, at pH 6.0 and 3.5 degrees C. A normal D2O solvent isotope effect (kcatH/kcatD = 1.5) is associated with the breakdown of the phosphoenzyme intermediate, indicating a solvent-derived proton in the transition state. The leaving group dependence of kcat/Km suggests that there is a strong electrophilic interaction between the enzyme and the leaving group oxygen in the transition state of the phosphorylation event. These results are compared with those of the Yersinia PTPase and suggest that the mechanism for PTPase-catalyzed phosphate monoester hydrolysis is conserved from bacterial to mammals.

Catalytic hydrolysis of phosphate monoesters by lanthanide(III) cryptate (2.2.1) complexes

The hydrolysis of 4-nitrophenyl phosphate is catalysed by lanthanide(III)(2.2.1) complexes, and kinetic studies reveal that the europium(III)(2.2.1) chloride (c= 4.0 mmol dm–3) hydrolyses the phosphate monoester at an enhanced rate (kp1= 1.7 × 10–4 s–1 at pH 7.5 and 50 °C), with a turnover number of 4; the pH dependence of the rate constants is well correlated with the concentration of the lanthanide (2.2.1) hydroxides.

Nature of the rate-determining steps of the reaction catalyzed by the Yersinia protein-tyrosine phosphatase.

Product inhibition and 18O exchange experiments suggest that the Yersinia protein-tyrosine phosphatase-catalyzed phosphate monoester hydrolysis proceeds through at least two different chemical steps, i.e. the formation and breakdown of a covalent phosphoenzyme intermediate. The pH dependence of kcat values is bell-shaped, with the apparent pKa derived from the acidic limb of the profile at 4.6 for both p-nitrophenyl phosphate and beta-naphthyl phosphate, whereas the apparent pKa derived from the basic limb of the profile is substrate-dependent, with apparent pKa values of 5.2 and 5.8 for p-nitrophenyl phosphate and beta-naphthyl phosphate, respectively. Twelve aryl phosphates with leaving groups having pKa values from approximately 7 to 10 are also examined as substrates at two pH values. At pH 4.0, the beta lg values is effectively zero, whereas at pH 7.5, a beta lg value of 0.16 is observed. Collectively, our results suggest that the rate-determining step under acidic conditions corresponds to the breakdown of the phosphoenzyme intermediate, whereas under more alkaline conditions, substrate effects also contribute to the rate-limiting step. A model is proposed for the mechanism of the Yersinia protein-tyrosine phosphatase-catalyzed reaction.

Phosphomonoesterase Enzymes That Utilize Histidine or Cysteine as Nucleophiles in SN2(P) Reactions

Abstract The past decade has seen a striking development in studies of the mechanisms used by a group of phosphomonoesterase enzymes that have been broadly defined as “acid” phosphatases on the basis of the pH optima that they exhibit with certain substrates. There is now convincing evidence that this apparent kinetic similarity masks the mechanistic use, by these enzymes, of at least two quite different nucleophilic residues in order to achieve catalysis in the hydrolysis of phosphate monoesters. The general properties of these enzymes are reviewed together with a summary of the extensive range of mechanistic information that is now available. This includes kinetic data that implicates the occurrence of a common rate limiting step, burst titration kinetics, stereochemical studies with phosphomonoesters that are chiral at phosphorus, trapping of covalent phosphoenzyme intermediates, and the identification of enzyme nucleophilic residues by covalent modification, trapping, spectroscopic and other experimental means. Finally, experiments are described that illustrate the use of active site-directed mutagenesis to explore the mechanisms of these enzymes. This research was supported by U. S. DHHS NIH Grant GM27003.

Phosphonate and Phosphate Monoester Hydrolysis

Abstract The reaction of phosphonate and phosphate monoesters with an epoxide yields β-hydroxyalkyl derivatives which upon treatment with base quantitatively release the original alcohol moiety of the ester in a relatively rapid simple procedure.

Catalytic hydrolysis of phosphate esters by metallocomplexes of 1,10‐phenanthroline derivatives in micellar solution

AbstractDivalent metal‐ion complexes of 2,9‐bis[(methyldodecylamino)methyl]‐1,10‐phenanthroline (C12PhenMII) in neutral Brij 35 micelles catalyse the hydrolysis of various phosphate triesters, diesters and monoesters. The catalytic activity of C12PhenMII has been compared with that of the metal‐ion complexes of its water‐soluble counterpart 2,9‐bis[(dimethyl‐amino)methyl]‐1,10‐phenanthroline (C1PhenMII). Saturation kinetics provide evidence for preliminary formation of ligand‐MII‐phosphate ester complexes, which decay to products. The hydrolysis of diphenyl 4‐nitrophenyl phosphate (1b) coordinated to C12PhenZnII proceeds 8700 times faster than the hydrolysis of 1b in the absence of metallocatalyst. Kinetic studies indicate that phosphate triesters containing a metal‐ion‐binding site in close proximity to the phosphoryl bond, i.e., diphenyl 5‐nitro‐2‐pyridyl phosphate (2b) and diphenyl 5‐nitro‐8‐quinolyl phosphate (3), are hydrolysed by the same mechanism as 1b.

Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions.

Formation of phosphate esters by kinases has long been recognized as an important process in biochemistry, but the reverse reaction, hydrolysis of phosphate esters by phosphatases, has attracted less attention. Recent work suggests that phosphatases are as important as kinases in regulatory processes, and that they constitute a diverse group of enzymes that utilize a variety of chemical means to accelerate phosphate ester hydrolysis.

Antibody-catalyzed hydrolysis of phosphate monoesters

Antibody-catalyzed hydrolysis of phosphate monoesters

Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase.

The guanidinium group of arginine-166 has been postulated to act as an electrophilic species during phosphorylation of alkaline phosphatase. Its role could be either to stabilize the developing negative charge on the oxygen of the leaving group or the pentacoordinate transition state or to help bind the -PO2-3 group. We have produced via site-directed mutagenesis two Escherichia coli alkaline phosphatase mutants (lysine-166 and glutamine-166) to test whether the guanidinium group plays a critical role in catalysis. Comparative kinetic characterization of the lysine-166 and glutamine-166 mutants indicates that the charge at residue 166 is not required for the hydrolysis of phosphate monoesters. Small decreases in kcat are observed for both the lysine and glutamine mutants, relative to the wild-type enzyme, but the value for the uncharged glutamine mutant is only one-third that of lysine. Thus, the stabilizing effect of the positively charged guanidinium group does not appear to play a major role in the rate-limiting step for substrate hydrolysis. A significant effect on the Km value is seen only for the glutamine mutant.

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.

Presteady State Kinetics of Phosphorothioate Hydrolysis by Alkaline Phosphatase: RATE-LIMITING DEPHOSPHORYLATION AT ALKALINE PH

Abstract The hydrolysis of O-p-phenylazophenylphosphorothioate by Escherichia coli alkaline phosphatase was studied by the stopped-flow technique. Burst kinetics is observed at both acid and basic pH, suggesting that a step following thiophosphorylation is rate-limiting at all pH values. At pH 8.5, activation of a second active site on the enzyme dimer is observed in the transient phase as substrate concentration is increased. The rate of the transient phase, however, decreases at high substrate concentration, suggesting that negative cooperativity exists between the two active sites. The implications of these results for the mechanism of hydrolysis of both O-phosphorothioate and phosphate monoesters by alkaline phosphatase are discussed.

Intramolecular general acid catalysis of phosphate monoester hydrolysis. The hydrolysis of salicyl phosphate

The effects of substituents in the 4- and 5-positions on the rate of hydrolysis of the salicyl phosphate dianion are entirely inconsistent with a mechanism involving a preliminary proton transfer to the leaving group. A mechanism is proposed in which the rate-determining step involves cleavage of the P–O bond concerted with a conformation change, but no significant amount of proton transfer. This mechanism accounts satisfactorily for all the known properties of the reaction, and predicts that intramolecular general acid catalysis by the carboxy-group in this system should be available for SN2(P) reactions also. It is found that the reactions of substituted pyridines with the salicyl phosphate dianion are indeed subject to catalysis, with rate enhancements as high as 108-fold.

Serum alkaline phosphatase.

The alkaline phosphatases constitute a large group of enzymes of low specificity that are involved in the hydrolysis of phosphate monoesters at a pH of about 9. They also play important roles in the transport of sugar and phosphate in the intestinal mucosa, kidney tubule, placenta, and bones. At the cellular level, they demonstrate activity against a wide variety of substrates but appear to be metabolically inert in the serum. Tissues that demonstrate the highest alkaline phosphatase activity include osteoblasts, bile canaliculi, intestinal mucosa, proximal convoluted tubules, and placenta. Alkaline phosphatase is present in serum, urine, feces, lymph, sweat, and breast milk. Large amounts are present in bile but whether the biliary tract represents the major mode of excretion still remains a matter of dispute. The serum alkaline phosphatase may originate then from bone, intestine, placenta, and the hepatobiliary system. Two possible mechanisms have been proposed to explain the elevated

The Serum Alkaline Phosphatase

The alkaline phosphatases constitute a large group of enzymes of low specificity that are involved in the hydrolysis of phosphate monoesters at a pH of about 9. They also play important roles in the transport of sugar and phosphate in the intestinal mucosa, kidney tubule, placenta, and bones. At the cellular level, they demonstrate activity against a wide variety of substrates but appear to be metabolically inert in the serum. Tissues that demonstrate the highest alkaline phosphatase activity include osteoblasts, bile canaliculi, intestinal mucosa, proximal convoluted tubules, and placenta. Alkaline phosphatase is present in serum, urine, feces, lymph, sweat, and breast milk. Large amounts are present in bile but whether the biliary tract represents the major mode of excretion still remains a matter of dispute. The serum alkaline phosphatase may originate then from bone, intestine, placenta, and the hepatobiliary system. Two possible mechanisms have been proposed to explain the elevated