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

Abstract

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, ...

Influence of Subunit Interface Mutations on Kinetic and Dynamic Properties of Alkaline Phosphatase from E. coli

“…31,32 Apparently negative cooperative kinetic behavior has been supported by a model describing an enzyme with inherently unequal subunits. 7 It has been proposed that enzyme asymmetry with subunits differing in affinity for the substrate and product, could be used to favor enhanced product release. An important feature of the proposed model is a conformational change converting a high affinity subunit to a low affinity subunit, and vice versa.…”

“…[1][2][3][4][5] It has been proposed that asymmetry of dimeric AP underlies such a kinetic behavior. 6 A plausible model based on subunit asymmetry, describing the advantages of homodimeric over monomeric enzymes, has been outlined. 7 Based on crystal structure analysis, 8 it has been anticipated that the β-pleated sheet, stretching from underneath the active site to the subunit interface, has an important role in conformational changes supporting the catalytic cycle.…”

“…6 A plausible model based on subunit asymmetry, describing the advantages of homodimeric over monomeric enzymes, has been outlined. 7 Based on crystal structure analysis, 8 it has been anticipated that the β-pleated sheet, stretching from underneath the active site to the subunit interface, has an important role in conformational changes supporting the catalytic cycle. The importance of interfacemediated communication between the subunits has been assessed by site directed mutagenesis.…”

Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli

“…3.1.3.1.) is one of many enzymes that display deviations from Michaelis-Menten kinetics resembling negative cooperativity [6,7]. A possible advantage of such a kinetic behaviour is not well understood.…”

Gene Cloning, Overproduction, and Characterization of Thermolabile Alkaline Phosphatase from a Psychrotrophic Bacterium

“…9) Of these APases, E. coli APase has been the most extensively studied for structures and functions. [10][11][12][13][14][15][16] This enzyme is located in the periplasmic space as a homodimer and is involved in the acquisition of phosphate from esters when free inorganic phosphate is depleted. Each subunit is composed of 449 amino acid residues and contains two Zn 2þ and one Mg 2þ ions.…”