A substrate-induced conformation change in the reaction of alkaline phosphatase from <i>Escherichia coli</i>

Halford, Stephen E.; Bennett, N. G.; Trentham, David R.; Gutfreund, H.

doi:10.1042/bj1140243

Cited by 77 publications

(57 citation statements)

References 15 publications

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“…For the native enzyme, the reported values for k4 (Scheme l) have ranged from 20 to 50 s -1 (19,44). This value is approximately the range reported in the literature for kcat, 13-?0 s -1, under a variety of conditions 09, 29,41,63). At acid pH, the rate-limiting step changes to dephosphorylation of the phosphoseryl intermediate, primarily because of a dramatic fall in the rate constant, k3, for this reaction from ~> 300 s-I (pH 8) to ~0.5 -1 (pH 5.5) 0 9).…”

Section: Limitingsupporting

confidence: 52%

“…(J. F. Chlebowski, personal communication). The enzyme hydrolyzes not only oxyphosphate monoesters (23,29,41,63), but also a variety of (58,59) and S-phosphorothioates (19), phosphoramidates (63), thiophosphate, and phosphate (3,19,65,69); hydrolysis of the latter group reflected by the catalysis of the exchange of 180 from H2180 into inorganic phosphate (3,65,69) or the release of H2S from thiophosphate (19). enzyme has an alkaline pH maximum, and the rate follows an approximately sigmoid pH-rate profile with an apparent pK, of ~ 7.5 (19,34,50,63).…”

Section: General Structure Of E Coli Alkaline Phosphatasementioning

confidence: 99%

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Structure and Mechanism of Alkaline Phosphatase

Coleman

1992

Annu. Rev. Biophys. Biomol. Struct.

814

448

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Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Limitingsupporting

confidence: 52%

Section: General Structure Of E Coli Alkaline Phosphatasementioning

confidence: 99%

Structure and Mechanism of Alkaline Phosphatase

Coleman

1992

Annu. Rev. Biophys. Biomol. Struct.

814

448

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“…A model describing the catalytic mechanism of APase from E. coli, based on the results of the kinetic experiments and in accordance with the data available in the literature, has been proposed. The model encompasses the experimental data indicating dimer asymmetry [19,26], unequal affinity of subunits for Mg 2+ and P i [6,9,20,24,25,28–31], conformational changes in the catalytic cycle [8,30,32–34], and the role of Mg 2+ in an allosteric activation. Asymmetry is an intrinsic characteristic of dimeric APase, and it is not the consequence of unequal saturation with Mg 2+ .…”

Section: Model Representation Of the Catalytic Cycle For Apase From Ementioning

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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“…However, the presteady-state transient phase at acid pH and the rate-limiting step at alkaline pH, which were shown to be mechanistically identical (14), had been assigned to the confomational change from E to E' (step 1 or 3) prior to phosphorylation by substrate. This suggestion was first made by Halford et al (15) prior to the discovery (12) of the endogenous phosphate found in preparations of alkaline phosphatase. Subsequently, influence of the Pi on the kinetics of the enzyme was rationalized as owing to its displacement of the E F= E' equilibrium.…”

Section: Alkaline Phssphatasementioning

confidence: 92%

Applications of ³¹P nuclear magnetic resonance to studies of protein structure and function

Sykes¹

1983

Can. J. Biochem. Cell Biol.

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Sykes, B. B. (1983) Applications of 3 '~ nuclear magnetic resonance to studies of protein structure and function. Can. J.Biochem. Cell Biol. 51, 155-1 6 4 Selective illustrative applications of phosphorus-3 1 nuclear magnetic resonance spectroscopy to the study of protein structure and function are reviewed, including the enzymes alkaline phosphatase, myosin, actin, and glycogen phosphorylase. Svkes, B. B. (1983) Applications of 3 '~ nuclear magnetic resonance to studies of protein structure and function. Can. J. Biochem. Cell Biol. 51, 155-1 6 4Revue des applications de la spectrsscopie de resonance magnktique nuclkaire du phosphore-31 B l'ktude de la structure et des fonction des proteines incluant les enzymes suivant: phosphatase alcaline, myosine, actine et glycogkne phosphorylase.[Traduit par la revue]?his statement refers to commonly used spin 1 /2 nuclei and, therefore, excludes nuclei such as tritium and thallium. Can. J. Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Alberta on 08/03/15For personal use only.

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A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

Cited by 77 publications

References 15 publications

Structure and Mechanism of Alkaline Phosphatase

Structure and Mechanism of Alkaline Phosphatase

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

Applications of ³¹P nuclear magnetic resonance to studies of protein structure and function

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A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

Cited by 77 publications

References 15 publications

Structure and Mechanism of Alkaline Phosphatase

Structure and Mechanism of Alkaline Phosphatase

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

Applications of 31P nuclear magnetic resonance to studies of protein structure and function

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Applications of ³¹P nuclear magnetic resonance to studies of protein structure and function