Mechanism of acylphosphatase inactivation by Woodward's reagent K (original) (raw)
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Common-type acylphosphatase: steady-state kinetics and leaving-group dependence
Biochemical Journal, 1997
A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pK a of the acids produced in hydrolysis) have been synthesized. The K m and V max values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 mC and pH 5.3. The values of k cat differ widely in relation to the different structures of the tested acyl phosphates : linear relationships between log k cat and the leaving group pK a , as well as between log k cat \K m and the leaving-group pK a , were observed. On the other hand, the K m values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme-substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol) ; nor
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
The present work compares the effects of several ligands (phosphatase substrates, MgCI2, RbCI and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37°C to 20°C and 0°C (hydrolysis experiments) or from 20°C to 0°C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmx for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Ka for the substrates; (ii) led to a 5-fold drop in the K a for the inorganic phosphate-induced di-occlnsion of E2(Rb); (iii) reduced the K0. s and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyi phosphate and acetyi phosphate and the Rb-promoted Ez(Rb) formation. At 20°C, in the presence of 1 mM RbCi and no Mg 2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgC! 2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCI in the incubation mixture. As a function of acetyl phosphate concentration the K m for Rh release was indistinguishable from the K m found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-uccluded fraction corresponding to maximal hydrolysis was not different from 10(O. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E 2 which has no K ÷ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na + + K +-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg 2÷ and K ÷ ions.
Journal of Biological Chemistry, 2001
Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H 2 O 2 at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and
Current Enzyme Inhibition, 2015
Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants can be derived from this plot: K max , K min , and K inflect. K max and K min represent the substrate to inhibitor concentration ratio for complete inhibition and minimal inhibition, respectively. K inflect represents the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to the inhibitory complex. These constants can be interpolated from the graph or calculated using the first and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot appears useful for known competitive and non-competitive inhibitors and may have general applicability.
The Contribution of Acidic Residues to the Conformational Stability of Common-Type Acylphosphatase
Archives of Biochemistry and Biophysics, 1999
Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, ⌬G(H 2 O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.
Properties of Cys21-mutated muscle acylphosphatases
Journal of Protein Chemistry, 1996
Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residueAn the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Set and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.
Biochemical Journal, 1980
The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysi...
Studies on the specificity of phosphorylase kinase using peptide substrates
The Journal of biological chemistry, 1977
The action of phosphorylase kinase on synthetic peptides is reported. These peptides are variants of the amino acid sequence. Ser-Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser-Val-Arg-Gly-Leu, found in the natural substrate, phosphorylase b. The effects of size, the cluster of basic groups at the NH2-terminal side, the phosphorylatable seryl residue, the hydrophobic groups surrounding serine, and the arginyl function at the COOH-terminal side were tested and analyzed by evaluation of the kinetic parameters, Km and Vmax. The first 6 residues were found to be nonessential, but substitution of residues in the sequence. Lys-Gln-Ile-Ser-Val-Arg, had a large effect on phosphorylation. A comparison was made between the action of nonactivated and activated phosphorylase kinase on selected peptides and phosphorylase b. Various forms of phosphorylase b were tested as substrates for cyclic AMP-dependent protein kinase in the presence of effectors and salts. Although phosphorylase would not serve as a su...
Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985
The prevailing conformations of partially purified pig kidney (Na++ K +)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb + or Ca 2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCI 2, 3 mM MnCI 2 or 1 mM CaCi2, the major enzyme conformation was E I. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCI 2. KCI, at 0.5 mM or 150 mM, produced an E 2 enzyme state; the effects of 0.5 mM KCI were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCI2/5 mM p-nitropbenyl phosphate/10 mM KCi) the (Na++ K+)-ATPase was in the E 2 state. At low ionic strength and 20°C and under 85% of maximal RbCI-stimulated phosphatase turnover (1 mM RbCI/3 mM MgCI2/5 mM p-nitrophenyi phosphate) no Rb + occlusion could be detected. Ca 2+, at low ionic strength and in the presence of 3 mM MgCI 2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained with 1 mM CaCI 2 were similar to those seen with 0.5 mM KCI; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca 2+ could not mimic Rb + in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na++ K +)-ATPase, the most abundant form is a non-occluding E 2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E 2 state.