Steady state kinetics and effect of SH inhibitors on acid phosphatase from bovine brain.

1. Product inhibition studies and transphosphorylation to methanol using two different substrates indicate that acid phosphatase from bovine brain forms a phosphoryl enzyme and that the phosphorylation step can not be rate limiting. 2. Acid phosphatase from bovine brain is inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid); this inhibition can be counteracted by inorganic phosphate. Incubation of the enzyme with p-nitrophenyl phosphate in the presence of p-chloromercuribenzoate leads, initially, to a higher degree of inhibition than that found with the same concentration of inhibitor in the absence of substrate. Both the titration by 5,5'-dithiobis-(2-nitrobenzoic acid) and inhibition by p-chloromercuribenzoate are consistant with the presence of 2 SH groups per mol of enzyme.     

Isolation of tau-phosphohistidine from a phosphoryl-enzyme intermediate of human prostatic acid phosphatase.

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.     

The catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase. pH and inhibition studies

Extensive kinetic studies of bovine intestinal 5'-nucleotide phosphodiesterase as a function of pH have confirmed and amplified the catalytic mechanism previously proposed on the basis of isolation of a covalent phosphorylated intermediate (Landt, M., and Butler, L.G. (1978) Biochemistry 17, 4130-4135). An enzyme-ionizing group with apparent pKa = 6.85 controls the rate-determining step. Electrostatic interactions between anionic substrate and two or more ionic groups on the enzyme have a major role in substrate binding. Binding of strongly inhibitory 5'-AMP is controlled by an ionizing group, probably on the enzyme, with pKa less than or equal to 5.9. At pH 6.0, imidazole is a classic uncompetitive inhibitor, in agreement with independent evidence that it stabilizes the covalent intermediate form of the enzyme. KI values for phosphonate analogs, which are competitive inhibitors, indicate that phosphodiesterase binds its products and product analogs more strongly than it binds substrate analogs. Some of the results presented here can be interpreted as indicating that 5'-nucleotide phosphodiesterase is the evolutionary precursor of alkaline phosphatase, with which it has many structural and catalytic properties in common, and which is found in relatively large amounts in the same tissue.     

Calcineurin-catalyzed reaction with phosphite and phosphate esters of tyrosine.

A convenient synthesis is reported for the preparation of the phosphite ester of tyrosine methyl ester. By use of calcineurin, at 30 degrees C, a phosphite ester was hydrolyzed with a VM value [119 nmol/(min.micrograms of E)] approximately 500 times greater than that obtained with tyrosine phosphate [0.23 nmol/(min.microgram of E)] as substrate, but with similar KM values (12 mM for Tyr-PH ME, 11 mM for Tyr-P). Acid phosphatase, on the other hand, hydrolyzed the phosphite ester with a VM and KM value lower than those obtained with tyrosyl phosphate. The temperature dependence of the kinetic parameters (KM and VM) was evaluated, and the activation parameters were obtained with both substrates. The entropy of activation associated with the enzymatic hydrolysis of tyrosine phosphate agrees with the entrophy change for the hydrolysis of the monoanion of phosphate monoesters. The energy of activation for both substrates was in agreement with the energy change for hydrolysis of the oxygen-phosphorous linkage of phosphate monoester monoanions and phosphite esters. These results are consistent with a scheme of general acid catalysis in the action of calcineurin.     

Effect of substitution inert metal complexes on calcineurin.

As a substitute for M(H2O)2+6, Co(NH3)3+6 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn2+, Mg2+, Co2+, and Co(NH3)3+6 were compared. Although kcat and Km were different with the metals, values of kcat/Km were nearly identical for Mn2+ and Mg2+, but lower for Co2+ and Co(NH3)3+6. The concentration of each metal providing half-maximal activation, designated Kact, was evaluated as 15.9 mM for Co(NH3)3+6, compared to Kact = 0.17 mM for Mn2+ and Co2+ and 6.3 mM for Mg2+, respectively. Comparing kcat/Kcat showed that Co(NH3)3+6 was a 170-fold poorer activator of calcineurin than was Mn2+, but only 1.5-fold poorer than Mg2+. Activation by Co(NH3)3+6 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH3)3+6 was found to support calcineurin activity, the related compound [Co-(ethylenediamine)3]3+ (or Co(en)3+3) was tested as a possible activator. Co(en)3+3 did not support calcineurin activity but did inhibit calcineurin. Co(en)3+3 showed competitive inhibition kinetics with either Mn2+ or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545-14550, 1986) and showed uncompetitive inhibition with Mn2+ as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal-substrate complex was not required for association with calcineurin.     

Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding. Br?nsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.     

Phosphotransferase activity associated with rat osseous plate alkaline phosphatase: a possible role in biomineralization.

1. Alkaline phosphatase from rat osseous plate catalyzed the transfer of phosphate from p-nitrophenylphosphate to glycerol, ethanolamines, Tris, glucose and 1-amino-1-methyl-2-propanol, in a wide range of pH. Serine did not stimulate phosphotransferase activity of the enzyme. 2. The best phosphotransferase acceptors were diethanolamine and glycerol while glucose was the poorest phosphotransferase acceptor used. 3. Diethanolamine and glycerol affected both VM and KM of p-nitrophenylphosphate hydrolysis with activation constants (KA) of 0.25 and 0.85 M, respectively. 4. A kinetic model was proposed for the phosphotransferase reaction observed with alkaline phosphatase from rat osseous plates.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

The pH dependence of binding of inhibitors to bovine adrenal tyrosine hydroxylase

The inhibition of purified bovine adrenal tyrosine hydroxylase by several product and substrate analogues has been studied to probe the kinetic mechanism. Norepinephrine, dopamine, and methylcatechol are competitive inhibitors versus tetrahydropterins and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine is an uncompetitive inhibitor versus tetrahydropterins and a competitive inhibitor versus tyrosine. The Ki value for 3-iodotyrosine depends on the tetrahydropterin used. These results are consistent with tetrahydropterin binding first to the free enzyme followed by binding of tyrosine. 5-Deaza-6-methyltetrahydropterin is a noncompetitive inhibitor versus tetrahydropterins and tyrosine. The effect of varying the concentration of tyrosine on the Ki value for 5-deaza-6-methyltetrahydropterin is consistent with the binding of this inhibitor to both the free enzyme and to an enzyme-dihydroxyphenylalanine complex. Dihydroxyphenylalanine also is a noncompetitive inhibitor versus tetrahydropterins and tyrosine; the effect of changing the fixed substrate is consistent with the binding of this inhibitor to both the free enzyme and to the enzyme-tetrahydropterin complex. The effect of pH on the Ki values was determined in order to measure the pKa values of amino acid residues involved in substrate binding. Tight binding of catechols requires that a group with a pKa value of 7.6 be deprotonated. Binding of 3-iodotyrosine involves two groups with pKa values of 7.5 and about 5.5, one of which must be protonated for binding. Binding of 5-deaza-6-methyltetrahydropterin requires that a group on the free enzyme with a pKa value of 6.1 be protonated. The Ki value for dihydroxyphenylalanine is relatively insensitive to pH, but the inhibition pattern changes from noncompetitive to competitive above pH 7.5, consistent with the measured pKa values for binding to the free enzyme and to the enzyme-tetrahydropterin complex.     

Competitive, uncompetitive, and mixed inhibitors of the alkaline phosphatase activity associated with the isolated brush border membrane of the tapeworm Hymenolepis diminuta

Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and beta-glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p-nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5'-adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (Ki') for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane-bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear.     

An innovative and cost-effective way to estimate alkaline phosphatase activity in in vitro cellular model systems

Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5. However, when this protocol is applied to the in vitro cellular model systems to estimate alkaline phosphatase activity, it tends to generate clumps of genomic DNA, leading to inaccurate pipetting for protein estimation. The aim of the study was to introduce minor modifications in the existing protocol to make it simple, cost-effective, with minimal labor-intensive procedures while estimating alkaline phosphatase activity in cellular model systems. The genomic DNA clumps were dissolved by depurination (adding 0.2 N HCl) and fragmentation (adding 0.2 N NaOH) during enzyme estimation. Moreover, these minor modifications have been standardized and optimized extensively by using serum samples (rich source of alkaline phosphatase), hFOB/ER9 (human Fetal osteoblastic cell) and HepG2 cells. Our results suggest that the modification incorporated in previously published method was robust enough to estimate ALP activity and protein concentration accurately. There was no significant variation in ALP activity estimated after modification (P > 0.05). This innovative approach could be beneficial for a researcher by providing an easy, cost effective and less labor-intensive solution for estimation of enzymatic activity in cellular model systems.     

Characterization of a phosphoenzyme intermediate in the reaction of phosphoglycolate phosphatase

When 32P-glycolate and phosphoglycolate phosphatase from spinach are mixed, 32P is incorporated into acid precipitated protein. Properties that relate the phosphorylation of the enzyme to the phosphatase are: the Km value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the 32P in the phosphoenzyme is diluted by unlabeled P-glycolate or the specific alternative substrate, ethyl-P; the activator Cl- enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of 32P-enzyme; and 32P is lost from the enzyme when 32P-glycolate is consumed. The phosphorylated protein has a molecular weight of 34,000, which is half that of the native protein and is similar in size to the labeled band that is seen on sodium dodecyl sulfate-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acylphosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by 32P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms (Rose, Z. B., Grove, D. S., and Seal, S. N. (1986) J. Biol. Chem. 261, 10996-11002).     

Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala (K(M) = 3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (Pi, Ki = 1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into Pi whether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone. an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis (Ki = 60 microM) and co-mediates 18O-label exchange into Pi in a concentration-dependent manner (K(M) = 64 microM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.     

Affinity-chromatography purification of alkaline phosphatase from calf intestine.

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.     

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.     

Effects of lead on K(+)-para-nitrophenyl phosphatase activity and protection by thiol reagents

Lead (Pb) inhibited K(+)-stimulated para-nitrophenyl phosphatase (K(+)-PNPPase) of rat brain P2 fraction in a concentration-dependent manner with IC50 3.5 microM. Altered pH versus activity demonstrated comparable inhibitions by Pb in buffered acidic, neutral and alkaline pH ranges. Inhibition of enzyme activity was higher at lower temperatures (17-27 degrees C) compared to 37 degrees C. Preincubation of enzyme with sulfhydryl (-SH) agents such as cysteine (Cyst) and dithiothreitol (DTT) but not glutathione (GSH) protected against Pb-inhibition. Uncompetitive type of inhibition with respect to the activation of K+ was indicated by a decrease in Vmax from 16.2 to 8.37 mumoles of para-nitrophenol (PNP)/mg protein/hr and Km from 18.99 to 12.39 mM. Kinetic studies on substrate (p-nitrophenyl phosphate) activation in the presence of Pb (3.5 microM) indicated a significant decrease in Vmax from 8.94 to 4.69 mumoles of PNP/mg protein/hr with no change in Km. Cyst (3 microM) and DTT (10 microM) reversed the Pb-inhibited Vmax from 4.69 to 8.38 and 7.24 mumoles of PNP/mg protein/hr respectively. These results suggest that the critical conformational property of K(+)-PNPPase is sensitive to Pb. The data also indicates that the Pb inhibits Na(+)-K+ ATPase system by interacting with dephosphorylation of the enzyme-phosphoryl complex, while Cyst and DTT protected against Pb-inhibition.     

Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen

The possibility of the existence of a covalent enzyme-phosphoryl intermediate, E-PO3, during catalysis of phosphate ester hydrolysis by the purple acid phosphatase (PAP) from bovine spleen has been examined. Transphosphorylation experiments show that up to 22% of the phosphoryl group from p-nitrophenyl phosphate (PNPP) can be transferred to primary alcohols. Burst experiments at high pH (9.1 or 8.1 for reduced or oxidized PAP, respectively), where hydrolysis of a phosphoenzyme intermediate is expected to be rate-limiting, show clear evidence for stoichiometric bursts of p-nitrophenolate from PNPP. The formation of base-stable, acid-sensitive adducts between PAP and the 32PO3 group of [gamma-32P]ATP has been demonstrated. The pH dependence of the kinetics parameters for reduced PAP has been determined over the range pH 3-8; a feature with a pKa of approximately 6.75 that is attributable to the enzyme-substrate complex is observed. Taken together, the present results are consistent with a two-stem pseudo Uni Bi mechanism that utilizes a covalent enzyme-phosphoryl intermediate, possibly a phosphohistidine.     

Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.     

Effects of metal elements on acid phosphatase activity in cucumber (Cucumis sativus L.) seedlings

Acid phosphatases (E.C.3.1.3.2) are a group of enzymes widely distributed in nature, which nonspecifically catalyze the hydrolysis of a variety of phosphate esters in pH ranges from 4 to 6 and play a major role in the supply and metabolism of phosphate in plants. The objective of the present study was to investigate the in vitro effects of some metals on the activity of acid phosphatase in cucumber seedlings (Cucumis sativus L.) and to determine their kinetic parameters. The enzyme was assayed with Hg, Cd, Mn, Pb, Zn, K and Na at the 0.001–1 mM range using ATP, PPi and β-glycerol phosphate as substrates. Mn, Na and Cd did not significantly alter the enzyme activity. K caused a broad activation at low concentrations and an inhibition at high concentrations (10 mM) and lead caused no inhibition. Acid phosphatase was inhibited by Hg and Zn and the inhibition type and IC₅₀ values were determined for these metals. Hg presented a mixed inhibition type with PPi and ATP as substrates and uncompetitive inhibition with β-glycerol phosphate as substrate. Zn presented competitive inhibition for ATP as substrate, and a mixed inhibition type with PPi and β-glycerol phosphate as substrate. IC₅₀ values were 0.02, 0.3 and 0.15 mM for Hg, and 0.056, 0.035 and 0.24 mM for Zn with ATP, PPi and β-glycerol phosphate as substrates, respectively. Analysis of these results indicates that Zn is a more potent inhibitor of acid phosphatase from cucumbers than Hg.     

Escherichia coli alkaline phosphatase. An analysis of transient kinetics

1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme-substrate complex.