Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli Stjepan Orhanovic ´ and Maja Pavela-Vranc ˇ ic ˇ Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, Croatia 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 [Orha- novic ´ S., Pavela-Vranc ˇ ic ˇ M. and Flogel-Mrs ˇ ic ´ M. (1994) Acta. Pharm. 44, 87–95]. 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, sug- gesting 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 con- trolled 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; sub- unit interactions; enzyme asymmetry; phosphate meta- bolism. Most unresolved questions, relating to the catalytic mech- anism of alkaline phosphatase (APase, E.C. 3.1.3.1), con- cern the influence of conformational changes and allosteric interactions on catalytic efficiency. Crystallographic ana- lysis 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 inter- actions 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 transphos- phorylation, 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. InthecaseofP i hydrolysis, phosphorylation of Ser102 is slow enough to become the rate-determining step [9]. APase activity increases in the presence of phosphate- accepting 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, the slow dissociation of P i ,andtheslow phosphorylation of Ser102 by P i , are both in accordance with P i binding in form of a trianion. The crystal structure of APase from E. coli has shown that metal–metal distances vary slightly between neighbor- ing subunits, but the significance of these differences is not clear. The Mg 2+ binding site is not close enough to allow for the direct participation of Mg 2+ in phosphomonoester hydrolysis [9]. The crystal structure of APase in complex with P i (APaseP i ), determined by Stec et al. differs from that resolved by Kim (1990), particularly with respect to the Ser102 conformation and the nature of the metal ion bound to the M3 site [10]. The APaseP i structure displays an increased mobility of the active site with pronounced anisotropy for the metal ions and the Arg166 side-chain [10]. APase belongs to a large group of enzymes displaying deviations from Michaelis–Menten kinetics, resembling negative cooperativity and Ôhalf-of-the-sitesÕ reactivity [11–15]. Although half-of-the-sites reactivity is a widespread phenomenon among oligomeric enzymes, a satisfactory explanation describing the advantage of such kinetic properties is still lacking [16,17]. Steady state kinetics, resulting in curved Lineweaver–Burk plots, did not agree Correspondence to M. Pavela-Vranc ˇ ic ˇ , Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, N. Tesle 12, 21000 Split, Croatia. Fax: + 385 21 385431, Tel.: + 385 21 385009, E-mail: pavela@pmfst.hr Abbreviations: APase, Alkaline phosphatase from E. coli; APaseP i , Alkaline phosphatase from E. coli containing inorganic phosphate; 2A2M1P, 2-amino-2-methyl-1-propanol; pNP, p-nitrophenol; p-NPP, p-nitrophenyl phosphate hexahydrate disodium salt. Enzymes: Alkaline phosphatase (PPB ECOLI, P00634), (E.C. 3.1.3.1.). (Received 2 July 2003, revised 4 September 2003, accepted 10 September 2003) Eur. J. Biochem. 270, 4356–4364 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03829.x with the flip-flop and half-of-the-sites mechanism [18]. In our previous work, APase from E. coli displayed deviations from Michaelis–Menten kinetics, producing concave (downwards) Hanes plots [19], the effect being more pronounced in the presence of a competitive inhibitor. Non-linear regression fitting, applied to equations descri- bing models based on either negative cooperative inter- actions between subunits or independent nonequivalent active sites, revealed that deviations in the presence of a competitive inhibitor could only be supported by a model assuming inherently nonequivalent subunits. The complex cooperative mode of metal-ion binding, resulting in unequal saturation of monomers with Mg 2+ , could lead to an in vivo dimer asymmetry. Therefore, the mode of activation with metal ions, as well as the dependence of the kinetic parameters and deviations from Michaelis–Menten kinetics on the Zn 2+ and Mg 2+ ion concentration, have been examined. APase could be used as a model enzyme to investigate the potential evolutionary advantage of homo- dimeric enzymes, having such kinetic properties, over a monomeric species. Here we present a model that describes the catalytic cycle of APase emphasizing the advantages that such a mechanism could have in conjunction to the proposed biological role of APase. Materials and methods Dialysis of the enzyme preparation APase from E. coli type III-S (Sigma Chemie GmbH, Taufkirchen, Germany) was dialyzed against three changes of 50 m M Tris/HCl (pH 8) containing 20 m M EDTA, followed by five changes of the same buffer without EDTA. Following dialysis, the protein concentration was deter- mined from the absorbance at 280 nm, using an absorption coefficient of e ¼ 0.72 M )1 Æcm )1 [20]. Metal free solutions The Zn 2+ ion concentration (2.7 · 10 )7 and 5 · 10 )7 M ) determined in distilled water and in 2-amino-2-methyl- 1-propanol (2A2M1P) buffer, respectively, was high enough to completely saturate all zinc binding sites in APase. In order to render the reaction mixture completely devoid of divalent metal ions, all solutions were prepared using distilled and deionized water, previously treated with an ion exchange resin (Chelex 100, Sigma, St. Louis, USA) with high specific affinity for divalent metal ions. Glassware was soaked prior to use in a mixture of H 2 SO 4 and HNO 3 (1 : 1, v/v), followed by washing in metal-free water. Chelex 100 was added to each buffer prior to pH adjustment. Enzyme activity, determined in metal-free reaction mixtures, com- prised 2–4% of the activity measured in the presence of sufficient Zn 2+ . Incubation in the presence of metal ions The enzyme solution was prepared by adding 15 lLof dialyzed enzyme to 750 lLof50m M Tris/HCl (pH 9). A ZnSO 4 and MgSO 4 solution (50 lL), of an appropriate concentration, was added to 51 lLoftheenzyme solution. Prior to measurement, the incubation mixture was placed for 23 h at 4 °C, followed by 1 h at room temperature. Spectrophotometric determination of the reaction rate The enzymatic activity was determined by measuring the absorbance change at k 405 nm and 25 °C, due to an increasing concentration of the reaction product, p-nitro- phenol (pNP), using the Lambda 40 Bio spectrophotometer (Perkin Elmer, Norwalk, USA). Activity was measured in a reaction mixture containing 2 mL of 0.35 M 2A2M1P buffer (pH 10.5), 50 lL of the enzyme solution and 50 lL of the substrate solution (p-nitrophenyl phosphate hexa- hydrate, disodium salt; pNPP) of an appropriate concen- tration in metal-free water. Kinetic analysis was performed using pNPP as substrate at concentrations ranging from 0.01 to 2 m M . Enzyme activation with Zn 2+ and Mg 2+ was followed using 2 m M pNPP. All reaction rate measurements were performed in duplicate. Curve-fitting procedure The kinetic parameters providing the best fit to the experimental data were determined using the nonlinear regression data analysis program, GRAFIT ,andtheHanes transformation of the equation developed for a model of an asymmetric enzyme [19]. Curves and kinetic constants, describing competitive inhibition, were obtained from respective data by applying the corresponding equation for competitive inhibition, using the kinetic parameters obtained without inhibitor as constants. The kinetic parameters are presented in Tables 1–5 along with the standard errors obtained by nonlinear regression analysis. The linearized transformation was applied, as the observed deviations from Michaelis–Menten kinetics were not readily detectable in the velocity vs. substrate concentration plot. Table 2. The affinity of subunit 1 and 2 for P i in dependence of the Zn 2+ to dimer ratio. Zn 2+ to dimer ratio K I1 (m M ) K I2 (m M ) 1.2 : 1 0.04 ± 0.004 0.19 ± 0.04 1.6 : 1 0.03 ± 0.004 0.12 ± 0.02 2 : 1 0.04 ± 0.004 0.27 ± 0.07 3.6 : 1 0.02 ± 0.003 0.13 ± 0.02 4 : 1 0.03 ± 0.01 0.10 ± 0.04 Table 1. The dependence of the kinetic parameters for APase from E. coli on the Zn 2+ to dimer ratio. Zn 2+ to dimer ratio K S1 (m M ) K S2 (m M ) V m (lmolÆmin )1 ) b 1.2 : 1 0.07 ± 0.02 1.76 ± 1.25 0.92 ± 0.14 1.02 ± 0.17 1.6 : 1 0.07 ± 0.02 1.21 ± 0.45 1.16 ± 0.27 1.63 ± 0.35 2 : 1 0.08 ± 0.01 1.72 ± 0.78 1.80 ± 0.22 1.41 ± 0.17 3.6 : 1 0.03 ± 0.01 1.57 ± 0.05 1.95 ± 0.02 1.54 ± 0.90 4 : 1 0.04 ± 0.04 1.96 ± 0.62 1.90 ± 0.42 1.79 ± 1.34 Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4357 Results Mode of metal ion activation, and the dependence of APase activity on the metal ion concentration In order to clarify the mode of APase activation by Zn 2+ , and to establish the appropriate Zn 2+ ion concentration in kinetic and Mg 2+ -activation experiments in 2A2M1P buffer at pH 10.5, enzymatic activity was determined at a Zn 2+ to dimer ratio ranging from 1 : 1 to 10 : 1. Figure 1 shows the dependence of the reaction rate on the Zn 2+ to dimer ratio. Enzymatic activity increases from 0.32, in the absence of Zn 2+ ,to7.26lmol pNPÆmin )1 in the presence of six Zn 2+ ions per dimer. A further increase of the Zn 2+ ion concentration to a Zn 2+ to dimer ratio of 8 : 1 and 10 : 1 reduces the enzymatic activity slightly. As the M3 site of native APase binds Mg 2+ [21], APase activation with Zn 2+ has also been followed in the presence of 2.1 · 10 )5 M Mg 2+ (Fig. 1). In the presence of Mg 2+ , a maximum activity of 9.05 lmolÆmin )1 pNP was attained at a Zn 2+ to dimer ratio of 4 : 1. A higher Zn 2+ to dimer ratio resulted in lower activity. The presence of Mg 2+ increases the catalytic efficiency of APase, although it appears that Mg 2+ is not directly involved in the catalytic step. The mechanism of APase activation by Mg 2+ is not fully understood. The influence of Mg 2+ could be limited to the subunit it binds to, or it could act on both subunits affecting the allosteric interactions and cooperativity that possibly exist between the subunits. Both phosphate-binding and calorimetric studies suggested posi- tive cooperativity of Zn 2+ binding to the M1 sites of the dimeric APase [5]. NMR studies indicate that metal ion migration from the M1 site of an inactive subunit to the M2 site of an active subunit is taking place [20,22]. The third and the fourth Zn 2+ probably do not bind to APase with the same affinity, whereas Mg 2+ binds to the M3 site with negative cooperativity [4–6,23]. Consequently, in the pres- ence of the substrate and Zn 2+ ions at a Zn 2+ to dimer ratio of 2 : 1, both ions bind to the same subunit, generating a dimer with only one active subunit. Therefore, Mg 2+ activation was studied using an enzyme fully saturated with Zn 2+ and having both subunits active, and an enzyme with two Zn 2+ ions bound to the dimer generating only one active subunit (Fig. 2). Table 4. The affinity of subunit 1 and 2 for P i in dependence of the Mg 2+ concentration at a Zn 2+ to dimer ratio of 2 : 1. [Mg 2+ ]( M ) K I1 (m M ) K I2 (m M ) – 0.04 ± 0.004 0.37 ± 0.10 2.1 · 10 )6 0.04 ± 0.001 0.20 ± 0.05 2.1 · 10 )5 0.04 ± 0.003 0.12 ± 0.01 2.1 · 10 )3 0.05 ± 0.003 0.74 ± 0.41 Table 5. The dependence of the kinetic parameters for APase from E. coli on the Mg 2+ concentration at a Zn 2+ to dimer ratio of 4 : 1. [Mg 2+ ] ( M ) K S1 (m M ) K S2 (m M ) V m (lmolÆmin )1 ) b – 0.04 ± 0.02 2.40 ± 2.63 1.90 ± 0.79 1.89 ± 0.66 2.1 · 10 )6 0.07 ± 0.02 0.64 ± 0.26 4.95 ± 1.30 1.19 ± 0.51 2.1 · 10 )5 0.07 ± 0.03 1.74 ± 1.88 5.58 ± 1.74 1.42 ± 0.42 2.1 · 10 )3 0.07 ± 0.01 1.18 ± 0.43 5.10 ± 0.71 1.16 ± 0.22 Table 3. The dependence of the kinetic parameters for APase from E. coli on the Mg 2+ concentration at a Zn 2+ to dimer ratio of 2 : 1. [Mg 2+ ] ( M ) K S1 (m M ) K S2 (m M ) V m (lmolÆmin )1 ) b – 0.08 ± 0.01 1.72 ± 0.78 1.47 ± 0.18 1.41 ± 0.17 2.1 · 10 )6 0.07 ± 0.01 2.56 ± 3.31 2.75 ± 0.41 1.11 ± 0.70 2.1 · 10 )5 0.08 ± 0.01 2.03 ± 0.53 3.05 ± 0.17 1.18 ± 0.07 2.1 · 10 )3 0.08 ± 0.02 2.51 ± 1.60 3.95 ± 0.54 1.52 ± 0.23 Fig. 1. Catalytic activity of APase from E. coli upon reactivation with Zn 2+ . The dialyzed enzyme was reactivated with Zn 2+ at varying Zn 2+ to dimer ratios in Tris/HCl (pH 9) in the absence of Mg 2+ (s), and in the presence of 2.1 · 10 )5 M Mg 2+ (h). Activity was deter- minedin0.35 M 2A2M1P buffer, pH 10.5, at 25 °Cusing2m M pNPP as substrate. Fig. 2. Semi-logarithmic plot of APase activity in dependence of the Mg 2+ concentration. Thedialyzedenzymewasreconstitutedwith Zn 2+ in Tris/HCl (pH 9) at a Zn 2+ to dimer ratio of 2 : 1 (h), and 4:1(s). The enzymatic activity was determined at varying Mg 2+ concentration in 0.35 M 2A2M1P buffer (pH 10.5) at 25 °Cusing 2m M pNPP as substrate. 4358 S. Orhanovic ´ and M. Pavela-Vranc ˇ ic ˇ (Eur. J. Biochem. 270) Ó FEBS 2003 Although Mg 2+ activates both Zn 2+ 2 APase and Zn 2+ 4 APase, the shape of the titration curve is fundament- ally different. The lowest Mg 2+ concentration used (0.001 m M ) almost completely activates Zn 2+ 4 APase, in contrast to the stepwise process of Zn 2+ 2 APase activation, demanding a significantly higher concentration of Mg 2+ (2.1 m M ). In the presence of a higher Mg 2+ concentration, the Zn 2+ 2 APase activity decreases sharply. A somewhat higher Mg 2+ concentration (over 4.2 m M )causesthe activity to drop for the Zn 2+ 4 APase enzyme. Influence of Zn 2+ on the kinetic parameters and the deviations from Michaelis–Menten kinetics A vast amount of data indicates that the subunits of the homodimeric APase from E. coli often do not display equal kinetic properties. It has been determined that P i binds to APase with negative cooperativity [6,8,9,24,25], the thermal inactivation has biphasic kinetics [26], and curve-fitting indicates that the deviations from Michaelis–Menten kine- tics are the consequence of unequal kinetic properties of the subunits [19]. It is possible that negative cooperativity in metal ion binding to the M3 site results in homodimer asymmetry. Consequently, the influence of Mg 2+ and Zn 2+ on the kinetic properties of APase and the deviations from Michaelis–Menten kinetics have been investigated. The kinetic properties have been determined for an enzyme reconstitutedwithanincreasingZn 2+ to dimer ratio in the absence (Fig. 3A), and in the presence of 0.05 m M P i (Fig. 3B). Deviations, present over the entire range of Zn 2+ concentrations examined, are apparently most pronounced at lower values. The kinetic constants, obtained using the curve-fitting procedure and describing the affinity of the subunits for the substrate (K S1 and K S2 )andforP i (K I1 and K I2 ), presented in Table 1 and Table 2, respectively, are independent of the Zn 2+ ion concentration. In order to support the conclusion that kinetic constants do not depend on the Zn 2+ concentration, curve-fitting was performed with a single constant value for each parameter (an average value for each kinetic constant was used) allowing only different V m values. There was no systematic deviation of the fit confirming that kinetic constants do not depend on the Zn 2+ concentration (results not shown). An increased Zn 2+ concentration results in higher V m values, while parameter b (determining the difference in the concentration and/or k cat of the subunits accommodating different conformations), does not change significantly in dependence of the Zn 2+ concentration. Influence of Mg 2+ on the kinetic properties of APase from E. coli Magnesium binds to the M3 site of native APase [1]. It activates the enzyme, but does not participate directly in phosphomonoester hydrolysis [3,4]. In the presence of Mg 2+ , the enzyme displays a higher V m at a constant K m value [6]. Due to negative cooperativity in metal ion binding to the M3 site, unequal saturation of the subunits with Mg 2+ could be the principal cause of conformational asymmetry of the homodimeric enzyme. Reaction mixtures with and without 0.05 m M P i ,ataZn 2+ to dimer ratio of 2 : 1 (Fig. 4A,B) and 4 : 1 (Fig. 5A,B), have been supple- mented with 2.1 · 10 )6 ,2.1· 10 )5 and 2.1 · 10 )3 M Mg 2+ . Deviations from linearity in the Hanes plot occur at all Mg 2+ concentrations examined. Deviations are apparently reduced in the presence of higher Zn 2+ and Mg 2+ concentrations, yet curve-fitting provides kinetic constants (K S1 , K S2 , b, K I1 and K I2 ), presented in Tables 3–6, that do not differ significantly for the metal ion concentrations tested. That conclusion was confirmed by successive curve- fitting with a single constant value for each parameter claimed to be independent of the Zn 2+ concentration (an average of all values determined for each experiment was used) allowing only V m to change (results not shown). Upon addition of Mg 2+ , V m gradually increases in reaction mixtures containing a lower Zn 2+ to dimer ratio. In the presence of a higher Zn 2+ to dimer ratio, V m approaches the maximum value even at the lowest Mg 2+ concentration tested. Increasing Zn 2+ and Mg 2+ concentrations do not affect the difference between the subunits with respect for their affinity for the substrate or the product (the difference Fig. 3. The influence of Zn 2+ on the kinetic properties of APase from E. coli. Catalytic activity was measured in 2A2M1P buffer, (pH 10.5) at 25 °CintheabsenceofP i (A) and in the presence of 0.05 m M P i (B) at a Zn 2+ to dimer ratio of 1.2 : 1 (.), 1.6 : 1 (n),2:1(d), 3.6 : 1 (s), and 4 : 1 (+). Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4359 between K S1 and K S2 ,andK I1 and K I2 , respectively). Also, parameter b is not significantly dependent on the metal ion concentration. It is noteworthy that the subunit with the highest affinity for the substrate almost has the same affinity for the product (the K I1 values are only slightly lower than the K S1 values), while the subunit with the lowest affinity for the substrate could bind P i more tightly (K I2 is considerably lower than K S2 ). Discussion Activation with Zn 2+ Maximum activity, achieved at a Zn 2+ to dimer ratio of 6 : 1 in the absence of Mg 2+ , is obtained when Zn 2+ is bound to the M1 and M2 site on both subunits and perhaps to one M3 site, that additionally activates the enzyme. An increased Zn 2+ ion concentration reduces the enzymatic activity indicating that binding of the last Zn 2+ ion, probably to the second M3 site, cannot supplement the role of magnesium in the kinetic mechanism. In the presence of Mg 2+ , maximum activity is accomplished at a Zn 2+ to dimer ratio of 4 : 1, probably resembling the form of the enzyme obtained with four Zn 2+ and one or two Mg 2+ ions bound [1,4]. Higher Zn 2+ concentrations decrease the enzymatic activity, probably by Zn 2+ binding to the magnesium binding site M3 [4]. Fig. 4. The influence of Mg 2+ on the kinetic properties of APase from E. coli. The influence of Mg 2+ on the kinetic properties of APase from E. coli inthepresenceofaZn 2+ to dimer ratio of 2 : 1 in 2A2M1P buffer, (pH 10.5) at 25 °CintheabsenceofP i (A), and in the presence of 0.05 m M P i (B). The reaction was followed in reaction mixtures containing either no Mg 2+ (+), or 2.1 · 10 )6 M ,(s); 2.1 · 10 )5 M , (d)and2.1· 10 )3 M ( · )Mg 2+ . Fig. 5. The influence of Mg 2+ on the kinetic properties of APase from E. coli. The influence of Mg 2+ on the kinetic properties of APase from E. coli at a Zn 2+ to dimer ratio of 4 : 1 in 2A2M1P buffer (pH 10.5) at 25 °C in the absence of P i (A), and in the presence of 0.05 m M P i (B). The reaction was followed in reaction mixtures containing either no Mg 2+ ,(+)or2.1· 10 )6 M ,(s); 2.1 · 10 )5 M , (d)and2.1· 10 )3 M (·)Mg 2+ . Table 6. The affinity of subunit 1 and 2 for P i in dependence of the Mg 2+ concentration at a Zn 2+ to dimer ratio of 4 : 1. [Mg 2+ ]( M ) K I1 (m M ) K I2 (m M ) – 0.04 ± 0.005 0.12 ± 0.04 2.1 · 10 )6 0.04 ± 0.002 0.11 ± 0.01 2.1 · 10 )5 0.05 ± 0.007 0.45 ± 0.19 2.1 · 10 )3 0.05 ± 0.008 0.42 ± 0.27 4360 S. Orhanovic ´ and M. Pavela-Vranc ˇ ic ˇ (Eur. J. Biochem. 270) Ó FEBS 2003 Activation with Mg 2+ The Mg 2+ -dependence of APase activity was examined with an enzyme reconstituted with Zn 2+ ions at Zn 2+ to dimer ratios of 2 : 1 and 4 : 1. As the activation experiments produced curves with fundamentally different shapes, it could be concluded that in the two reaction mixtures APase occurs in a different form. At a Zn 2+ to dimer ratio of 2 : 1, due to positive cooperativity in Zn 2+ binding [5] and migration of a metal ion from the M1 site of the inactive subunit to the M2 site of an active subunit [20,22], the enzyme is expected to be present in the form containing two Zn 2+ ions on the same monomer. Also, the different shapes of the curves indicate that the mode of Mg 2+ activation is not the same for Zn 2+ 2 APase as for Zn 2+ 4 APase. The more pronounced activity increase with Zn 2+ 4 APase is probably due to the influence of Mg 2+ in an allosteric interaction. A higher Mg 2+ concentration is necessary for a successive activation of Zn 2+ 2 APase, because the dimer with only one active subunit cannot display allosteric interactions. Hence, a slow activation could result from the generation of an enzyme with Zn 2+ at both M1 sites and Mg 2+ in the M2 site characterised by almost normal transphosphorylating activity but considerably lower hydrolytic activity [9,27]. Lower Zn 2+ 2 APase and Zn 2+ 4 APase activity, in the presence of a high Mg 2+ concentration, is probably due to Mg 2+ binding to the zinc binding sites (M2 and M1). It appears that in contrast to the binding of Zn 2+ to the second M3 site, Mg 2+ binding in the range of Mg 2+ concentrations examined (if it binds at all due to negative cooperativity) does not reduce the enzymatic activity. Deviation from linearity in the dependence on the Zn 2+ ion concentration Deviations from linearity will depend on the difference between the subunits in their affinity for the substrate (difference between K S1 and K S2 ), and on parameter b describing the difference in V m between the subunits. Deviations will be more pronounced if parameter b is large and if the subunit affinities differ widely. An increase in the Zn 2+ concentration is followed only by an increase in V m with the remaining kinetic parameters not changing con- siderably. According to the kinetic parameters, deviations from Michaelis–Menten kinetics are not reduced in the presence of higher Zn 2+ concentrations. In the Hanes plot, deviations are apparently reduced as an increased V m reduces the slope of the curve, making the deviations less obvious. Analysis was performed by normalization of all curves to the same V m to verify that deviations did not depend on the Zn 2+ concentration as judged from the kinetic constants. The curves normalized by V m were superimposable with equally obvious deviations for all Zn 2+ concentrations (results not shown). Deviations from Michaelis–Menten kinetics were observed in the presence of low Zn 2+ concentrations that cannot generate a fully metal- saturated dimer. This implies that interactions between the subunits are not responsible for the observed deviation. Therefore, the cause of non-Michaelis–Menten kinetics could only be due to a mixture of subunits differing in conformation and catalytic properties. Parameter b does not change depending on the Zn 2+ concentration, thus, indicating that Zn 2+ does not influence the equilibrium concentration of the subunits. Deviation from linearity in the dependence of the Mg 2+ ion concentration It has been determined that the affinity of the subunits for the substrate and the product does not depend on the Mg 2+ concentration. Curves normalized to the same V m show the same deviations for all Mg 2+ concentrations employed (results not shown). An increased Mg 2+ concen- tration gradually activates the enzyme when partially saturated with Zn 2+ , while the fully saturated enzyme almost instantaneously achieves maximum activity at the lowest Mg 2+ concentration tested. Such a mode of activa- tion suggests that Mg 2+ facilitates allosteric interactions in an enzyme with four Zn 2+ ions bound. Parameter b does not show any regular dependence on the Mg 2+ concentra- tion. Had negative cooperativity in Mg 2+ binding induced the dimer asymmetry, deviation from linearity would have been most pronounced in the presence of an Mg 2+ concentration that saturates only one subunit. As deviations are present in the reaction mixture devoid of Mg 2+ , it could be concluded that Mg 2+ does not induce APase asymmetry. Parameter b does not depend on the Mg 2+ concentration, indicating that Mg 2+ equally enhances catalysis of both subunits. Model representation of the catalytic cycle for APase from E. coli 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 experi- mental 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. Asym- metry is an intrinsic characteristic of dimeric APase, and it is not the consequence of unequal saturation with Mg 2+ . The difference in stability of the conformationally different subunits is apparently not large, allowing for the existence of a conformationally heterogeneous mixture of subunits even in the presence of the Zn 2+ ion concentration saturating only one monomer. The homodimer could become asymmetric because of negative cooperativity in ligand binding. The respective ligand can be an amino acid side-chain from the active site region, leading to homo- dimer asymmetry. It has been established that Ser102, the amino acid acting as a primary nucleophile in the active site of APase from E. coli, could adopt two conformations in a dimer saturated with P i [10]. The proposed model (Scheme 1) assumes that subunit 1 displays high affinity for both the substrate and the product, while subunit 2 binds the ligand with considerably lower affinity. Because of a high affinity for the product, subunit 1 has a low k cat , in contrast to subunit 2 showing a lower affinity for the product and consequently a higher k cat . In the presence of a low substrate concentration, subunit 1 is predomin- antly active (reaction path A). An increased substrate Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4361 concentration activates the second subunit following reaction path B and C. In the presence of a low substrate concentration, phos- phomonoester hydrolysis proceeds via reaction path A. The high affinity subunit 1 binds the substrate molecule and a covalent intermediate is formed accompanied by alcohol dissociation. Upon hydrolysis, P i slowly dissociates from the high affinity subunit. Higher substrate concentrations activate reaction path B and C. The APase dimer, with P i bound to the high affinity subunit, binds the substrate molecule to the low affinity subunit. In reaction path B, all reactions take place on the subunit with lower affinity, while in reaction path C, the first event is the interchange of subunit conformations. After a conformational change, P i dissociates easily from the low affinity subunit, leaving the substrate tightly bound to the high affinity subunit. Reaction path B describes a mechanism with subunit 2 completely independent of subunit 1, with no conforma- tional changes taking place in the course of the catalytic cycle. Substrate binding to subunit 2 could be followed by a conformational change transforming dimer 12 into 21, as described in reaction path C. The kinetic constants K I1 and K I2 , describing the affinity for P i ,differlessthan constants K S1 and K S2 . Therefore, the dimer with the substrate bound to the high affinity subunit (21) is more stable than the dimer with the product bound to the subunit with higher affinity (12). It facilitates product release, and prevents substrate dissociation. Following the conformational change, the product could easily dissociate from subunit 2, while the substrate remains bound to subunit 1 for a new catalytic cycle. The constants K S1 , K I1 and V m describe reaction path A with one active subunit, while constants K S2 , K I2 and b describe the kinetic properties of paths B and C with both subunits active. The advantage of an asymmetric dimer, over a mono- meric species, would be the additional possibility of enhanced or conformationally controlled product release. The crystal structure and the reaction mechanism of APase from E. coli, suggested by Kim and Wyckoff [1], as well as the high resolution crystal structure determined by Stec et al. [10], offers clarification of the subunit affinity differences at a molecular level. The crystal structure determined in the presence of P i indicates that both substrate (phosphomonoester) and product (P i )bindin thesamewaytotheactivesite[1].Therefore,theenzyme with high affinity for the substrate also has a high affinity for the product. The reaction product, P i , is probably bound with even higher affinity, due to the influence of Zn 2+ in the M2 site. It is known that the enzyme is more easily phosphorylated with a phosphomonoester than with P i , and that the product of the transphosphorylation reaction dissociates much faster than P i [9,27]. It has been suggested that a possible reason for such a difference may be the binding of P i as a trianion [9]. Perhaps the trianion cannot be avoided because its generation is enhanced by the same catalytic Zn 2+ ion involved in the formation of the nucleophile for the hydrolysis of the covalent inter- mediate. Alternatively, the mechanism that includes the trianion may have evolved in order to control the dissociation of the valuable product, P i . Therefore, some kind of a mechanism must have evolved either to prevent trianion formation, or to utilize it as a kinetic switch for controlled product release. It is probable that the active site adopts a new conforma- tion in order to separate P i from Zn 2+ occupying the M2 site. The APaseP i conformation, described by Stec et al. [10], with a Zn 2+ replacing Mg 2+ in the M3 site and the side- chain of Ser102 removed from the phosphate binding site, could represent the conformation of the subunit allowing product dissociation. As the side-chain of Ser102 is hydro- gen bonded to Thr155 at an increased distance from the catalytic Zn 2+ ion, this conformation could not be effective in phosphomonoester hydrolysis. If the crystal structure determined by Stec et al. [10] resembles the conformation of subunit 2, reaction path B is not possible. APase could catalyze phosphomonoester hydrolysis with a high k cat but only via reaction path C that involves a conformational change from a 12- to a 21-dimer. As an altered Ser102 conformation does not necessarily change the affinity for the substrate or the product, it is likely that the altered geometry of an active site prevents formation of a trianon. The reaction velocity should depend on the frequency of the conformational change from 12 to 21, which will depend on the concentration of the substrate inducing such a change. The same conformational change could be induced or enhanced by any ligand with a different binding affinity for subunits 1 and 2. If the ligand concentration is higher than that of the substrate, the conformational change occurs more often, enhancing the overall reaction velocity. The catalytic path A, active in the presence of low substrate concentrations, could be enhanced in the same way. Both activation of APase with Mg 2+ and kinetic data indicate that Mg 2+ enhances the reaction rate influencing allosteric Scheme 1. The reaction cycle of APase from E. coli. High affinity subunit 1 (h); low affinity subunit 2 (s); covalently bound inorganic phosphate (-P); phosphomonoester (ROP); alcohol (ROH). 4362 S. Orhanovic ´ and M. Pavela-Vranc ˇ ic ˇ (Eur. J. Biochem. 270) Ó FEBS 2003 interactions in the reaction mechanism of APase from E. coli. It has been established that Mg 2+ binds to APase with negative cooperativity [6,21]. It increases the reaction rate, while it does not affect the affinity for the substrate. According to the crystal structure, the subunit containing Mg 2+ has a higher affinity for the substrate (corresponding to subunit 1), and binds the substrate in a way that enables catalysis. Inorganic phosphate formed upon hydrolysis of the covalent intermediate, remains bound to subunit 1 until subunit 2 binds the substrate or Mg 2+ (Scheme 2). The subunit with higher affinity for P i has a higher affinity for Mg 2+ also. Mg 2+ binds to the low affinity subunit enhancing the conformational change in path C, and enabling a conformational change in path A, thereby increasing the rate of both cycles. In reaction path A, binding of Mg 2+ to subunit 2 induces a conformational change from 12 to 21. Inorganic phos- phate and Mg 2+ dissociate from the low affinity subunit, while the neighboring high affinity subunit can easily bind another substrate molecule. In reaction path C, the second Mg 2+ binds to subunit 2 following substrate binding. It enhances a conformational change inducing the release of the product and Mg 2+ , thereby leaving an Mg 2+ ion and a molecule of the substrate bound to the subunit capable of catalyzing hydrolysis. Therefore, binding of Mg 2+ in a negatively cooperative fashion to the M3 site of dimeric APase increases the rate of the conformational change responsible for the activation of the enzyme. Conforma- tionally controlled product dissociation could enhance metabolite transfer to another protein as the conformational change could be facilitated by an interaction with an acceptor protein or a transmembrane channel. In case of APase it would allow simultaneous diffusion of Mg 2+ and P i into the cell. It has been shown that the PiT transport system for P i in E. coli cotransports P i and Mg 2+ [35]. Acknowledgements This work was supported by a grant from the Croatian Ministry of Science and Technology Nr. 177050. References 1. Kim, E.E. & Wyckoff, H.W. (1990) Structure of alkaline phos- phatases. Clin. Chim. Acta. 186, 175–187. 2. Anderson, R.A. & Vallee, B.L. (1975) Cobalt (III), a probe of metal binding sites of Escherichia coli alkaline phosphatase. Proc. NatlAcad.Sci.USA72, 394–397. 3. Anderson, R.A., Bosron, W.F., Kennedy, F.S. & Vallee, B.L. (1975) Role of magnesium in Escherichia coli alkaline phospha- tase. Proc. Natl Acad. Sci. USA 72, 2989–2993. 4. Bosron, W.F., Anderson, R.A., Falk, M.C., Kennedy, F.S. & Vallee, B.L. (1977) Effect of magnesium on the properties of zinc alkaline phosphatase. Biochemistry 16, 610–614. 5. Chlebowski, J.F., Mabrey, S. & Falk, M.C. (1979) Calorimetry of alkaline phosphatase. Stability of the monomer and effect of metal ion and phosphate binding on dimer stability. J. Biol. Chem. 254, 5745–5753. 6. Cathala, G. & Brunel, C. (1975) Bovine kidney alkaline phos- phatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg 2+ stimulation. J. Biol. Chem. 250, 6046–6053. 7. Barrett, H., Butler, R. & Wilson, I.B. (1969) Evidence for a phosphoryl-enzyme intermediate in alkaline phosphatase cata- lyzed reactions. Biochemistry 8, 1042–1047. 8. Gettins, P. & Coleman, J.E. (1983) 31 P nuclear magnetic resonance of phosphoenzyme intermediates of alkaline phosphatase. J. Biol. Chem. 258, 408–416. 9. Coleman, J.E. (1992) Structure and mechanism of alkaline phos- phatase. Annu. Rev. Biophys. Biomol. Struct. 21, 441–483. 10. Stec, B., Holtz, K.M. & Kantrowitz, E.R. (2000) A revised mechanism for the alkaline phosphatase reaction involving three metal ions. J. Mol. Biol. 299, 1303–1311. 11. Bernhard, S.A., Dunn, M.F., Luisi, P.L. & Schack, P. (1970) Mechanistic studies on equine liver alcohol dehydrogenase. I. The stoichiometry relationship of the coenzyme binding sites to the catalytic sites active in the reduction of aromatic aldehydes in the transient state. Biochemistry 9, 185–192. 12. Lazdunski, M., Petitclerc, C., Chappelet, D. & Lazdunski, C. (1971) Flip-flop mechanisms in enzymology. A model: the alkaline phosphatase of Escherichia coli. Eur. J. Biochem. 20, 124–139. 13. Levitzki, A., Stallcup, W.B. & Koshland, D.E. Jr (1971) Half- of-the-sites reactivity and the conformational states of cytidine triphosphate synthetase. Biochemistry 10, 3371–3378. 14. Stallcup, W.B. & Koshland, D.E. Jr (1973) Half-of-the sites reactivity and negative co-operativity: the case of yeast glycer- aldehyde 3-phosphate dehydrogenase. J. Mol. Biol. 80, 41–62. 15. Stallcup, W.B. & Koshland, D.E. Jr (1973) Half-of-the sites reactivity in the catalytic mechanism of yeast glyceraldehyde 3-phosphate dehydrogenase. J. Mol. Biol. 80, 77–91. 16. Ward, W.H. & Fersht, A.R. (1988) Tyrosyl-tRNA synthetase acts as an asymmetric dimer in charging tRNA. A rationale for half- of-the-sites activity. Biochemistry 27, 5525–5530. 17. Ward, W.H. & Fersht, A.R. (1988) Asymmetry of tyrosyl-tRNA synthetase in solution. Biochemistry 27, 1041–1049. Scheme 2. The reaction cycle of APase from E. coli in the presence of Mg 2+ . High affinity subunit 1 (h); low affinity subunit 2 (s); cova- lently bound inorganic phosphate (-P); phosphomonoester (ROP); alcohol (ROH). Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4363 18. Waight, R.D., Leff, P. & Bardsley, W.G. (1977) Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase. Biochem. J. 167, 787–798. 19. Orhanovic ´ , S., Pavela-Vranc ˇ ic ˇ ,M.&Flogel-Mrs ˇ ic ´ , M. (1994) Evidence for asimmetry of alkaline phosphatase from E. coli. Acta Pharm. 44, 87–95. 20. Gettins, P. & Coleman, J.E. (1983) 113 Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases. J. Biol. Chem. 258, 396–407. 21. Coleman, J.E., Nakamura, K. & Chlebowski, J.F. (1983) 65 Zn(II), 115 Cd(II), 60 Co(II), and Mg(II) binding to alkaline phosphatase of Escherichia coli. Structural and functional effects. J. Biol. Chem. 258, 386–395. 22. Otvos, J.D. & Armitage, I.M. (1980) Determination by cadmium- 113 nuclear magnetic resonance of the structural basis for metal ion dependent anticooperativity in alkaline phosphatase. Bio- chemistry 19, 4031–4043. 23. Lazdunski, C., Petitclerc, C., Chappelet, D., Leterrier, F., Douzou, P. & Lazdunski, M. (1970) Tight and loose metal binding sites in the apoalkaline phosphatase of E. coli. Reconstitution of the Ca 2+ -phosphatase from the apoenzyme EPR study of the Mn 2+ -phosphatase. Biochem. Biophys. Res. Commun. 40, 589–593. 24. Applebury, M.L., Johnson, B.P. & Coleman, J.E. (1970) Phos- phate binding to alkaline phosphatase. Metal ion dependence. J. Biol. Chem. 245, 4968–4976. 25. Chappelet-Tordo, D., Iwatsubo, M. & Lazdunski, M. (1974) Negative cooperativity and half of the sites reactivity. Alkaline phosphatases of Escherichia coli with Zn 2+ ,Co 2+ ,Cd 2+ ,Mn 2+ , and Cu 2+ in the active sites. Biochemistry 13, 3754–3762. 26. Malhotra, O.P., Singh, L.R. & Srivastava, D.K. (1983) Molecular asymmetry in alkaline phosphatase of Escherichia coli. Arch. Biochem. Biophys. 220, 519–529. 27. Trentham, D.R. & Gutfreund, H. (1968) The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli. Biochem. J. 106, 455–460. 28. Bosron, W.F., Kennedy, F.S. & Vallee, B.L. (1975) Zinc and magnesium content of alkaline phosphatase from Escherichia coli. Biochemistry 14, 2275–2282. 29. Coleman, J.E. & Gettins, P. (1983) Alkaline phosphatase, solution structure, and mechanism. Adv. Enzymol. Relat. Areas. Mol. Biol. 55, 381–452. 30. Cathala, G. & Brunel, C. (1975) Bovine kidney alkaline phos- phatase. Purification, subunit structure, and metalloenzyme properties. J. Biol. Chem. 250, 6040–6045. 31. Sun, L., Kantrowitz, E.R. & Galley, W.C. (1997) Room tem- perature phosphorescence study of phosphate binding in Escheri chia coli alkaline phosphatase. Eur. J. Biochem. 245, 32–39. 32. Halford, S.E., Bennett, N.G., Trentham, D.R. & Gutfeund, H. (1969) A substate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli. Biochem. J. 114, 243–251. 33. Schlyer, B.D., Schauerte, J.A., Steel, D.G. & Gafni, A. (1994) Time-resolved room temperature protein phosphorescence: non- exponential decay from single emitting tryptophans. Biophys. J. 67, 1192–1202. 34. Subramaniam, V., Bergenhem, N.C., Gafni, A. & Steel, D.G. (1995) Phosphorescence reveals a continued slow annealing of the protein core following reactivation of Escherichia coli alkaline phosphatase. Biochemistry 34, 1133–1136. 35. van Veen, H.W. (1997) Phosphate transport in prokaryotes: molecules, mediators and mechanisms. Antonie Van Leeuwenhoek 72, 299–315. 4364 S. Orhanovic ´ and M. Pavela-Vranc ˇ ic ˇ (Eur. J. Biochem. 270) Ó FEBS 2003 . Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli Stjepan Orhanovic ´ and Maja Pavela-Vranc ˇ ic ˇ Department of. representation of the catalytic cycle for APase from E. coli A model describing the catalytic mechanism of APase from E. coli, based on the results of the kinetic