Phosphate binding to Escherichia coli alkaline phosphatase. Evidence for site homogeneity (original) (raw)
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European Journal of Biochemistry, 1970
The labelling of the Zn2+ and Co2+-phosphatase with [32P]pyrophosphate was studied a t different pH. The formation of complexes between arsenate and the enzyme has been investigated using a variety of techniques : kinetics, equilibrium dialysis, isolation of the complex, determination of its activity for [32P]pyrophosphate. The mechanism of the alkaline phosphatase is obviously very peculiar. Its main features are: (a) Only one of the two sites can be phosphorylated a t alkaline pH from [32P]pyrophosphate in the Zn2+ as in the Co2+-phosphatase. Both sites are reactive a t acidic pH. (b) The binding of two arsenate molecules to the Co2+-phosphatase a t pH 8.0 has an anticooperative character which we found previously in 1969 for the binding of orthophosphate to the Zn2+ phosphatase. (c) The incubation of the enzyme a t pH 8.5 in high concentrations of [74As]arsenate gives rise to a complex containing 2 moles of arsenate per mole of dimer. One C74As]arsenate is loosely bound and easily exchangeable for unlabelled arsenate ; the other [74As]arsenate is very firmly bound and not exchangeable. A detailed study of this complex suggests that it is an hybrid complex containing one covalently bound and one noncovalently bound arsenate. One never observes the formation of a diarsenylated enzyme.
Biochemistry, 1974
Evidence for the nonequivalence of the active sites in dimeric alkaline phosphatase of Escherichia coli has been obtained both at acidic and alkaline pH with Zn2+, Co2+, Cu2+, and Cd2+ enzymes. (1) Reaction of all metallophosphatases with 2,4-dinitrophenyl phosphate proceeds through a transient phase (a burst) at pH 4.1 and 5". A biphasic burst of about 2 mol of dinitrophenol/mol of dimer was obtained with Zn2+, Co2+, Cu2+, and Cd2+ phosphatases. The biphasicity of the burst indicates that the first site is phosphorylated more rapidly than the second site. The first part of the burst (1 mol of phenol/mol of enzyme) is too fast to be followed by the stopped-flow technique with Co2+ and Cu2+ phosphatases (kol
Biochemistry, 1993
Sitqrspecific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the K, values for both enzypes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. 'The Ki for phosphate, which is pH-and Mg2+-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that a t pH 8.0 the hydrolysis of the covalent enzymephosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis. The introduction of either the D153H or the D153H/K328H mutations reduces the heat stability of the enzyme, whereas the K328H mutation alone exhibits the same heat stability as the wild-type enzyme. Energy minimization and Langevin molecular dynamics calculations suggest that the enhanced phosphate affinity of the D153H enzyme is due to the repositioning of Lys-328 so that it can directly interact with the phosphate and thereby stabilize its binding to the enzyme. These data also indicate that although Asp-153 is not a direct ligand to Mg2+ in the wild-type enzyme, it plays a role in stabilizing the binding of Mg2+ in the M3 site and indicates that the Mg2+ at the M3 site is important for catalysis by stabilizing the active conformation of the enzyme. The introduction of histidine residues a t both position 153 and 328 results in an enzyme that is remarkably similar to mammalian alkaline phosphatases in terms of pH versus activity profile, heat stability, maximal activity, and stimulation by Mg2+, suggesting that these two mutations are responsible for many of the differences in properties between the E. coli and mammalian alkaline phosphatases. Alkaline phosphatase (EC 3.1.3.1) is a nonspecific phosphomonoesterase that functions through a phosphoseryl intermediate (Schwartz & Lipmann, 1961) to produce inorganic phosphate and an alcohol. In the presence of a phosphate acceptor such as ethanolamine or Tris, the enzyme catalyzes a transphosphorylation reaction with the transfer of the phosphoryl group to the alcohol (Dayan & Wilson, 1964; Wilson et al., 1964). The catalytic mechanism has been the subject of numerous kinetic (Coleman & Gettins, 1983) and structural studies (Kim & Wyckoff, 1989; Kim & Wyckoff, 1991). The rate-determining step of the mechanism is pH dependent; at acidic pH the hydrolysis of thecovalent enzymephosphate complex (E-P) is rate-limiting, while under basic conditions the rate-limiting step becomes the release of phosphate from the noncovalent enzyme-phosphate complex (E-Pi) (
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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1991
The effects of K +, Na + and nucleotides (ATP or ADP) on the steady-state phosphorylafion from [3ZP]P i (0.5 and | mM) and acetyi [a~P]phosphate (AeP) (5 raM) were studied in membrane fragments and in proteofiposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20°C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K + and to 20% by 10 mM K + plus I mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K + but was 30% incre&sed by 50 mM Na+. In proteofiposomes E-P Naext, (ii) about 50% reduced by 5, 10 or 100 formation from Pi was (i) not influenced by 5-10 mM K~ or 100 mM + m]VI Ke+t and (iii) completely prevented by 50 mM + Na,~. Enzyme phosphorylation from AcP was 30% increased by 10 K~ or 50 mM Na,wt; these E-P were 50% redneed by 10-100 mM Kex t. However, E-P formed from AcP without mM 4. 4. 4-Key t or Nac~ t was not affected by extraceHular K4-. nuor~ence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E z state in the presence of 10 mM Na 4-or 2 mM K 4-but to an El state in the absence of both cations. With pNPP, the data indicated an E~ state in the absence of Na + and K 4. and also in the presence of 20 mM Na+, and an E 2 form in the presence of 5 raM K 4-. These results suggest that, although with some slmHarities, the reversible P~ phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.