Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase.

Anderson, Richard A.; Vallée, Bert L.

doi:10.1073/pnas.72.1.394

Cited by 31 publications

(22 citation statements)

References 17 publications

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“…Successful loading of SECs was observed with four different enzymes; a-amylase (56 kDa), 40 b-galactosidase (540 kDa), 41 sHRP (100 kDa) 42 and ALP (89 kDa). 43 The a-amylase was stained with Evans blue and encapsulated in SECs in order to be observed by LSCM (see Fig. 2e).…”

Section: Encapsulation Of Proteinsmentioning

confidence: 99%

Viability of plant spore exine capsules for microencapsulation

et al. 2011

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Sporopollenin exine capsules (SECs) (outer exoskeletal wall of the spores of Lycopodium clavatum) were extracted and examined for their potential use as microcapsules. They were shown, by laser scanning confocal microscopy (LSCM), to be void of their inner contents. The removal of nitrogenous and other internal materials was supported by a combination of elemental and gravimetric analyses. Two different methods were investigated to encapsulate substances into SECs which were (i) mild passive migration of materials into the SECs and (ii) subjecting SECs and materials to a vacuum. A range of fluorescent dyes with different polarities were seen using LSCM to encapsulate efficiently into the SECs (up to 1 g.g À1 ). Relatively unstable materials with different polarities were encapsulated into the SECs: polyunsaturated oils, which are labile to oxidation, and the enzymes streptavidin-horseradish peroxidase (sHRP) and alkaline phosphatase (ALP). Irrespective of the encapsulation techniques employed no oxidation of the oils or denaturation of the enzymes was observed following their full recovery. This study gives the first indication of the viability of SECs to microencapsulate various potentially unstable materials without causing a detrimental effect.

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Section: Encapsulation Of Proteinsmentioning

confidence: 99%

Viability of plant spore exine capsules for microencapsulation

et al. 2011

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“…Crystallographic analysis has shown that APase from E. coli has three metal binding sites [1]. Both zinc ions in the active site are essential for activity [2], whereas magnesium alone does not activate the apoenzyme but increases the activity of the Zn 2+ ‐containing APase [3,4]. Significant cooperative interactions have been detected during metal‐ion binding, positive for the binding of Zn 2+ to the M1 site, and negative for the binding of the activating cations to the M3 site [5,6].…”

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confidence: 99%

Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli

Orhanović¹,

Pavela-Vrančić²

2003

European Journal of Biochemistry

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Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits [Orhanovic´S., Pavela-VrancˇicˇM. and Flogel-Mrsˇic´M. (1994) Acta. Pharm. 44,[87][88][89][90][91][92][93][94][95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg 2+ binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analyses. An activation study has indicated that Mg 2+ enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn 2+ or Mg 2+ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release. APase displays higher activity in the presence of Mg 2+ , as binding of Mg 2+ increases the rate of conformational change. A conformationally controlled and Mg 2+ -assisted dissociation of the reaction product (P i ) could serve as a kinetic switch preventing loss of P i into the environment.Keywords: metalloenzymes; conformational change; subunit interactions; enzyme asymmetry; phosphate metabolism.Most unresolved questions, relating to the catalytic mechanism of alkaline phosphatase (APase, E.C. 3.1.3.1), concern the influence of conformational changes and allosteric interactions on catalytic efficiency. Crystallographic analysis has shown that APase from E. coli has three metal binding sites [1]. Both zinc ions in the active site are essential for activity [2], whereas magnesium alone does not activate the apoenzyme but increases the activity of the Zn 2+ -containing APase [3,4]. Significant cooperative interactions have been detected during metal-ion binding, positive for the binding of Zn 2+ to the M1 site, and negative for the binding of the activating cations to the M3 site [5,6]. Phosphomonoester hydrolysis and transphosphorylation, catalyzed by APase, proceeds through a covalent serine-phosphate intermediate [7,8]. Dissociation of the reaction product, P i , is rate limiting at alkaline pH.In the case of P i hydrolysis, phosphorylation of Ser102 is slow enough to become the rate-determining step [9]. APase activity increases in the presence of phosphateaccepting alcohols. The rate of P i formation is unchanged, indicating that the newly generated phosphomonoester dissociates much faster than P i . It has been suggested that P i is bound to the active site in form of a dianion [9], however, ...

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“…It can also be obtained from Escherichia coli, which has similar catalytic properties, similar pH-rate profile, and forms the same phosphoseryl intermediate as the intestinal enzyme [33]. The molecular weight is 89 kDa for E. coli ALP [34] and 126-140 kDa for bovine intestinal ALP [32][33][34][35]. ALP exists as a dimmer comprising two very similar or identical subunits each containing 429 amino acids [33].…”

Section: Enzymesmentioning

confidence: 99%