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Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme.

ABSTRACT: 1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P(i) to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

SUBMITTER: Halford SE

PROVIDER: S-EPMC1178530 | biostudies-other | 1972 Mar

REPOSITORIES: biostudies-other

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Studies on alkaline phosphatase. Transient-state and steady-state kinetics of Escherichia coli alkaline phosphatase.

Project description:1. The transient-state and steady-state phases of the reaction between Escherichia coli alkaline phosphatase and 4-methylumbelliferyl phosphate were investigated by using a fluorimetric stopped-flow technique. 2. At low substrate concentration (5mum) in the pH range 3.8-6.3 there was an initial rapid liberation of up to 1mole of 4-methylumbelliferone/mole of enzyme. 3. At very low substrate concentration (0.1mum) in the pH range 4.9-5.9 an initial lag in 4-methylumbelliferone production was observed, from which values for k(+1) and k(-1) could be obtained. 4. The pH profiles for the rates of phosphorylation and dephosphorylation are quite different, and it is postulated that an ionizing group which determines the conformation during the phosphorylation step is not involved in the dephosphorylation step. 5. The binding constants for substrate and P(i) are similar throughout the pH range 4-8. The ionization of substrate or P(i) appeared to have no marked effect on the binding.

| S-EPMC1187806 | biostudies-other

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

Project description:Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62-242 milliunits/mg and phosphate ester hydrolysis with specific activities of 41-61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.

| S-EPMC419532 | biostudies-literature

Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase.

Project description:1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis-Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/nu) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis-Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.

| S-EPMC1183727 | biostudies-other

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Project description:Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.

| S-EPMC7513700 | biostudies-literature

Optimization, Characteristics, and Functions of Alkaline Phosphatase From <i>Escherichia coli</i>.

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| S-EPMC8899610 | biostudies-literature

Potent Alkaline Phosphatase Inhibitors, Pyrazolo-Oxothiazolidines: Synthesis, Biological Evaluation, Molecular Docking, and Kinetic Studies.

Project description:To develop new alkaline phosphatase inhibitors (ALP), a series of pyrazolo-oxothiazolidine derivatives were synthesized and biologically assessed, and the results showed that all of the synthesized compounds significantly inhibited ALP. Specifically, compound 7g displayed the strongest inhibitory activity (IC50 = 0.045 ± 0.004 μM), which is 116-fold more active than monopotassium phosphate (IC50 = 5.242 ± 0.472 μM) as a standard reference. The most potent compound among the series (7g) was checked for its mode of binding with the enzyme and shown as non-competitively binding with the target enzyme. The antioxidant activity of these compounds was examined to investigate the radical scavenging effect. Moreover, the MTT assay method was performed to evaluate their toxic effects on the viability of MG-63 human osteosarcoma cells, and all compounds have no toxic effect on the cells at 4 μM. Computational research was also conducted to examine the binding affinity of the ligands with alkaline phosphatase, and the results revealed that all compounds showed good binding energy values within the active site of the target. Therefore, these novel pyrazolo-oxothiazolidine derivatives might be employed as promising pharmacophores for potent and selective alkaline phosphatase inhibitors.

| S-EPMC9654533 | biostudies-literature

Escherichia coli alkaline phosphatase. An analysis of transient kinetics.

Project description: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.

| S-EPMC1178056 | biostudies-other

Escherichia coli alkaline phosphatase. Relaxation spectra of ligand binding.

Project description:The temperature-jump technique was used to study the binding equilibrium between the Escherichia coli alkaline phosphatase dimer and 2-hydroxy-5-nitrobenzyl phosphonate in 0.1m-tris buffer, pH8.0. Three partially discrete relaxations were observed, two of which could be related to the bimolecular associations of ligand with different conformations of the enzyme and the third to the interconversion of these states. Relaxation spectra were also used to analyse the changes in the mechanism of ligand binding to alkaline phosphatase caused by increase in ionic strength. The relaxation spectrum observed after the addition of P(i) to the equilibrium mixture of phosphonate and enzyme was also studied. Difference spectroscopy indicated that both of these ligands were bound to the alkaline phosphatase dimer at the same time. These results are related to the catalytic mechanism of this enzyme, with particular reference to the role of two identical subunits in a dimeric enzyme that exhibits only one active site functioning in catalysis at any given time.

| S-EPMC1178432 | biostudies-other

Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli.

Project description:Inhibition of siderophore biosynthetic pathways in pathogenic bacteria represents a promising strategy for antibacterial drug development. Escherichia coli synthesize and secrete the small molecule iron chelator siderophore, enterobactin, in response to intracellular iron depletion. Here we describe a detailed kinetic analysis of EntE, one of six enzymes in the enterobactin synthetase gene cluster. EntE catalyzes the ATP-dependent condensation of 2,3-dihydroxybenzoic acid (DHB) and phosphopantetheinylated EntB (holo-EntB) to form covalently arylated EntB, a product that is vital for the final assembly of enterobactin. Initial velocity studies show that EntE proceeds via a bi-uni-uni-bi ping-pong kinetic mechanism with a k(cat) equal to 2.8 s(-1) and K(m) values of 2.5, 430, and 2.9 microM for DHB, ATP, and holo-EntB-ArCP, respectively. Inhibition and direct binding experiments suggest that, during the first half-reaction (adenylation), DHB binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), phosphopantetheinylated EntB binds to the enzyme followed by the release of products, AMP and arylated EntB. Two hydrolytically stable adenylate analogues, 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) and 5'-O-[N-(2,3-dihydroxybenzoyl)sulfamoyl]adenosine (DHB-AMS), are shown to act as slow-onset tight-binding inhibitors of the enzyme with (app)K(i) values of 0.9 and 3.8 nM, respectively. Direct binding experiments, via isothermal titration calorimetry, reveal low picomolar dissociation constants for both analogues with respect to EntE. The tight binding of Sal-AMS and DHB-AMS to EntE suggests that these compounds may be developed further as effective antibiotics targeted to this enzyme.

| S-EPMC2860672 | biostudies-literature

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli.

Project description:The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.

| S-EPMC1995121 | biostudies-literature

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