Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase.
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ABSTRACT:
SUBMITTER: Trotman CN
PROVIDER: S-EPMC1177109 | biostudies-other | 1971 Aug
REPOSITORIES: biostudies-other
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Project description:Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.
| S-EPMC3171187 | biostudies-literature
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
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
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
Project description:Little is known about how metalloproteins in the secretory pathway obtain their metal ion cofactors. We used the Pho8 alkaline phosphatase of the yeast Saccharomyces cerevisiae to probe this process in vivo. We found that both Pho8 activity and protein accumulation are zinc-dependent and decrease in zinc-limited cells. Low Pho8 accumulation was the result of degradation by vacuolar proteases. Surprisingly, the protective effect of zinc on Pho8 stability was not solely due to Zn(2+) binding to the active-site ligands suggesting that the Pho8 protein is targeted for degradation in zinc-limited cells by another mechanism. Pho8 appears to be a rare example of a metalloprotein whose stability is regulated by its metal cofactor independently of active-site binding. We also assessed which zinc transporters are responsible for supplying zinc to Pho8. We found that the Zrc1 and Cot1 vacuolar zinc transporters play the major role while the Msc2/Zrg17 zinc transporter complex active in the endoplasmic reticulum is not involved. These results demonstrate that the vacuolar zinc transporters, previously implicated in metal detoxification, also deliver zinc to certain metalloproteins within intracellular compartments. These data suggest that Pho8 receives its metal cofactor in the vacuole rather than in earlier compartments of the secretory pathway.
| S-EPMC2741694 | biostudies-literature
Project description:The amino acids involved in the coordination of two Zn2+ ions and one Mg2+ ion in the active site are well conserved from EAP (Escherichia coli alkaline phosphatase) to BIAP (bovine intestinal alkaline phosphatase), whereas most of their surrounding residues are different. To verify the consequences of this heterology on their specific activities, we compared the activity and structure recoveries of the metal-free forms (apo) of EAP and of BIAP. In the present study, we found that although the sensitivities of EAP and BIAP to ions remained similar, significant differences in dimeric structure stability of apo-enzymes were observed between EAP and BIAP, as well as in the kinetics of their activity and secondary structure recoveries. After mild chelation inactive apo-EAP was monomeric under mild denaturing conditions, whereas inactive apo-BIAP remained dimeric, indicating that the monomer-monomer contact was stronger in the mammalian enzyme. Dimeric apo-EAP (0.45 microM, corresponding to 4 units/ml) recovered approx. 80% of its initial activity after 3 min incubation in an optimal recovery medium containing 5 microM Zn2+ and 5 mM Mg2+, whereas dimeric apo-BIAP (0.016 microM, corresponding to 4 units/ml) recovered 80% of its native activity after 6 h incubation in an optimal recovery medium containing 0.5 microM Zn2+ and 5 mM Mg2+. Small and different secondary structure changes were also observed during activity recoveries of apo-BIAP and apo-EAP, which were not in parallel with the activity recoveries, suggesting that distinct and subtle structural changes are required for their optimal activity recoveries.
| S-EPMC1316277 | biostudies-literature
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
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
Project description: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.
| S-EPMC1178530 | biostudies-other
Project description:To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only approximately 12% wild-type activity as measured by k(cat(amine)). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60-90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.
| S-EPMC2817917 | biostudies-literature