Mechanistic Insight from Calorimetric Measurements of the Assembly

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Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes Marcelo M. Pedroso, Fernanda Ely, Margaret C. Carpenter, Natasa Mitic, Lawrence R Gahan, David Louis Ollis, Dean E. Wilcox, and Gerhard Schenk Biochemistry, Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 7, 2017

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Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacer aerogenes Marcelo M. Pedroso,a Fernanda Ely,a Margaret C. Carpenter,b Nataša Mitić,c Lawrence R. Gahan,a David L. Ollis,d Dean E. Wilcox,b* Gerhard Schenka*

a

School of Chemistry and Molecular BioSciences, The University of Queensland, St Lucia, QLD 4072, Australia; b Department of Chemistry, Dartmouth College, Hanover, NH 03755, United States of America; c Department of Chemistry, National University of Ireland - Maynooth, Maynooth, Co. Kildare, Ireland; d Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.

Funding Source Statement: This work was financially supported by the Australian Research Council, Discovery Projects Scheme (DP150104358); MMP was supported by an International Postgraduate Research Scholarship and a University of Queensland International Living Allowance Scholarship; DEW thanks the National Science Foundation (USA) for financial support from grant CHE1308598; NM thanks the Science Foundation of Ireland for financial support in the form of a President of Ireland Young Researcher Award (SFI-PIYRA).

To whom correspondence should be addressed: Gerhard Schenk ([email protected]) Tel.: 61-7-33654144, Fax: 61-7-33654273, and Dean Wilcox ([email protected]) Tel.: 1-603-6462874, Fax: 1-603-6463946

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Abbreviations. CI, condition independent; EDTA, ethylenediaminetetracetic acid; EEC, enthalpy-entropy compensation; EPR, electron paramagnetic resonance; GpdQ, glycerophosphodiesterase from Enterobacter aerogenes; ITC, isothermal titration calorimetry; MCD, magnetic circular dichroism.


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Abstract Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its β site until substrate is present. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the β site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the β site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first and second coordination sphere residues at the β site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.


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Hydrolysis of biological molecules is catalyzed by different classes of enzymes, with some using the acid-base chemistry of intrinsic residues and others employing the Lewis acidity of one or more metal ions to increase the electrophilicity of the substrate and/or nucleophilicity of water.1 Some metallohydrolases have two tightly bound metal ions (e.g., urease (Ni),2 alkaline phosphatase (Zn),3 arginase (Mn)4), while others have one tightly bound metal ion and a second that binds with the substrate (e.g., methionine aminopeptidase (Co),5 certain metallo-β-lactamases (Zn)6).7 The role of each metal ion is of considerable importance, not only to elucidate the enzymatic mechanism but for therapeutic applications, such as inhibiting metallo-β-lactamases associated with antibiotic resistance.8 Proposed mechanisms for these enzymes typically include a metalbound hydroxide nucleophile that is stabilized by the metal ions in the active site.9 Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a classic α/β-sandwich fold and a hexameric quaternary structure (trimer of dimers).10 It is distantly related to other bimetallic hydrolases, such as purple acid phosphatases, Ser/Thr protein phosphatases and the 3`-5` cyclic nucleotide diesterase Rv0805 from Mycobacterium tuberculosis,11-13 and it has gained recent attention as a potential bioremediator due to its ability to degrade the byproducts from hydrolysis of the nerve agent VX.14 Several divalent transition metal ions, including Zn(II), Co(II), Mn(II), Cd(II) and Fe(II), support the catalytic activity of GpdQ.15-17 Kinetic, crystallographic and spectroscopic data reveal that the resting state of the enzyme is predominately an inactive mononuclear form with one metal ion bound at the six-coordinate α site (Figure 1).15-18 A second metal ion binds to the β site in the presence of substrate, an example of substratepromoted formation of a catalytically competent active site. For example, addition of the competitive inhibitor and substrate analogue phosphate led to a modest increase in the affinity of the α site for Mn(II), with Kd dropping from 29 µM to 17 µM, but a significant (10-fold) increase in the affinity of the β site for Mn(II).16 Substrate binding also induces the slow dissociation of one of the ligands of the β metal ion, Asn80, leading to fully active enzyme, an example of coordination flexibility to optimize catalytic efficiency.16 While substrate-promoted formation of a catalytically competent binuclear site may be beneficial for cellular function, it limits the potential use of GpdQ in 4 ACS Paragon Plus Environment

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bioremediation. Thus, an important goal is to develop mutants of the enzyme that maintain catalytic activity independent of the presence of substrates. Replacing Asn80 by an aspartate (Figure 1) leads to an increase in the metal affinity of the β site, but the catalytic efficiency (kcat/KM) decreases significantly from ~16 s-1mM-1 to ~6 s-1µM-1.15,16 However, substituting an alanine for the second coordination sphere residue Ser127, which forms a hydrogen bond with Asn80 (Figure 1), leads to a slight increase in the catalytic efficiency and a modest increase in the affinity of the β site for metal ions.19 This Ser127Ala mutant of GpdQ has been immobilized on magnetic nanoparticles to demonstrate its ability to degrade organophosphates over an extended period of time.20 Since there is an intimate connection between the activity of GpdQ and its affinity for metal ions, we have used isothermal titration calorimetry (ITC) to quantify the Co(II) and Mn(II) affinities and, in particular, the metal binding thermodynamics of the wildtype protein and the Asn80Asp and Ser127Ala mutants, both in the absence and presence of phosphate. Because much of the available structural and functional data for GpdQ have been determined with Co(II),15-18 it was used predominantly in this study, although titrations with Mn(II) were included for comparison to the Co(II) results and to Mn(II) binding constants obtained earlier with EPR spectroscopy.16 Fortuitously, the difference in the metal affinities of the α and β sites of wild-type GpdQ and the two mutant proteins allow the binding thermodynamics of each site to be quantified separately and sequential formation of the catalytic binuclear site to be investigated. Most importantly, the ability of ITC to quantify proton displacement upon metal binding has provided new insight about formation of the hydroxide nucleophile, which is shown to accompany binding of the first metal ion to the GpdQ active site.

Experimental Procedures Escherichia coli BL21(DE3) and DH5α host cells were purchased from Novagen. All chromatographic devices (FPLC system, chromatographic protein standards and relevant resins) were purchased from GE Healthcare, while all chemicals and buffers were purchased from Sigma Chemical Co. The protease inhibitor cocktail was purchased from Roche.


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The GpdQ-encoding expression vectors pCY76::GpdQ for the wild-type protein and for the Asn80Asp and Ser127Ala mutants were used to transform competent E. coli DH5α cells,15-21 which were grown in 4 L of TB medium that contained 50 µg/mL ampicillin and 0.1 mM CoCl2 for 36 hours at 30 ˚C. Identical procedures were used to obtain all three protein samples. The cells were then harvested by centrifugation and lysed using a French Press at 1000 psi. The soluble fraction was loaded onto a HiPrep 16/10 DEAE column, equilibrated with 20 mM Tris.HCl buffer, pH 8.0, and the proteins were eluted with a linear 0-1 M NaCl gradient. Fractions containing activity against 2 mM bis-para-nitrophenyl phosphate were dialyzed against 20 mM HEPES buffer, pH 8.0, containing 1.5 M (NH4)2SO4. The dialysate was then loaded onto a HiLoad 26/10 Phenyl Sepharose column, equilibrated with 20 mM HEPES, pH 8.0, containing 1 M (NH4)2SO4. Fractions containing phosphodiesterase activity were concentrated to approximately 4 mL and loaded onto a HiPrep 16/10 Sephacryl S-200 gel filtration column, equilibrated with 20 mM HEPES, pH 8.0, containing 0.15 M NaCl. The GpdQ concentration was measured at 280 nm using ε280= 39,880 M-1.cm-1 (monomeric unit).15,21 A typical purification yielded approximately 30 mg of protein per liter of medium, with activity similar to that reported earlier.15,16 Apo protein of wild-type GpdQ and the Asn80Asp and Ser127Ala mutants was obtained by incubating approximately 3 mg of protein in a 3 mL solution containing 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM 1,10-phenanthroline, 5 mM 2,6pyridinedicarboxylic acid, 5 mM 8-hydroxyquinone-5-sulfonic acid and 5 mM 2mercaptoethanol in 20 mM HEPES, pH 7.0, at 4 °C. After 24 hours of incubation the protein was separated from the chelating solution using an Econo-Pac 10DG gel filtration column equilibrated with the desired buffer that had been treated previously with Chelex® resin. The absence of metal ions in the protein solutions was confirmed by atomic absorption spectroscopy. All ITC measurements were obtained with a MicroCal iTC200 at 25 °C, unless indicated otherwise, using the apo form of wild-type and mutant GpdQ proteins in the 50250 µM (typically 120-170 µM) concentration range in 50 mM buffer, pH 7.25. Injection volumes were 1.0 µL (initial 0.2 µL injection not included in the analysis) with 150-600 sec between injections as needed to ensure complete binding with each aliquot. For 6 ACS Paragon Plus Environment

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comparison with most ITC literature, the data are reported in calories. Titrations were carried out using either MnCl2 or CoSO4 dissolved in 50 mM buffer, pH 7.25, and were performed in the absence or the presence of 10 equivalents of K2HPO4, relative to the protein concentration. For the latter experiments, both the enzyme and metal ion solutions contained the same concentration of the phosphate salt. Prior to measurements, the ITC sample cell was soaked with a saturated solution of EDTA for an hour, washed with Milli-Q® water and rinsed with buffer. The concentrations of the metal ion solutions were verified with standardized EDTA solutions and atomic absorption spectroscopy. At least three sets of data were collected for each experiment and used to determine the average experimental values. The ITC data were fitted using MicroCal Origin® 7.0 software that uses a nonlinear algorithm that minimizes the χ2 value by fitting the heat flow associated with each injection to an equation corresponding to an equilibrium binding model.22 The heat of dilution, which was obtained from the end of the titration, was subtracted from the integrated data, and a mathematical model of one (3 parameters) or two (6 parameters) independent binding sites was used to fit the integrated data. Derivations of the mathematical models used for the data fitting are described elsewhere.23,24 The best-fit values provide the average experimental binding stoichiometry (nITC), change in enthalpy (∆HITC) and binding constant (KITC) for each site. Since coupled solution chemistry of the metal ions (e.g., metal-buffer interactions) and protons (buffer (de)protonation) contribute to the experimental binding enthalpy and binding constant, a post hoc analysis of these values is necessary to determine the number of protons that are displaced from, or bind to, the protein and then the condition-independent (CI) binding affinity (Kd) and binding thermodynamics (∆G°CI, ∆HCI, ∆SCI). The data were analyzed as described elsewhere.2528

The entropic contribution to the binding free energy is indicated by the value –T∆SCI,

which has the same sign convention and units as the enthalpic contribution, ∆HCI.

Results The ITC data for Mn(II) binding to apo wild-type GpdQ consist of exothermic peaks with a single inflection, and similar exothermic data with a larger amount of heat are observed for Co(II) binding (Figure 2). The integrated titration data were fitted using 7 ACS Paragon Plus Environment

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a one-site binding model and Table 1 summarizes the average experimental best-fit values. The binding stoichiometry is approximately one metal ion per protein monomer. No clear evidence is observed for a second metal ion binding, due to a low affinity and/or negligible net heat. The protein binds the first Co(II) at the α site about an order of magnitude more tightly than it does Mn(II) (KITC ~106 and ~105, respectively). As shown in Figure 3, Mn(II) or Co(II) binding to GpdQ in the presence of phosphate is also exothermic but more complex. The integrated ITC data now have two sequential inflections and were fitted with a two independent sites binding model, which gives stoichiometries of approximately one metal ion per binding site. The presence of phosphate increases the affinity of the α site for Mn(II) by ~30-fold and its affinity for Co(II) over 70-fold (Table 1). When metal ions bind to a protein they often displace protons from metalcoordinating residues or alter the pKa of nearby residues, resulting in the loss of protons.23,24 For metallohydrolases, formation of a metal-stabilized hydroxide nucleophile would also result in the loss of a proton. The number of displace protons can be determined from a series of ITC titrations in buffers with different enthalpies of protonation (e.g., HEPES, -5.0 kcal/mol; Tris, -11.3 kcal/mol) and the contribution of these protons to the net enthalpy from their binding to the buffer.25,27 This analysis of the enthalpy for titrations of Co(II) into GpdQ in different buffers at pH 7.25, both in the absence and the presence of phosphate (Figure S1), shows that 1.1 protons are displaced from the α site in the absence of phosphate, and 1.0 and 0.2 are displaced from the α and β sites, respectively, in the presence of phosphate. To determine the buffer-independent binding thermodynamics, contributions to the experimental binding constant and binding enthalpy from coupled equilibria involving buffer protonation by displaced protons and buffer competition for the metal ion were subtracted in a post hoc analysis.25-28 This analysis provides the conditionindependent (CI) thermodynamic values found in Table 1. The change in heat capacity (∆Cp°) associated with binding can be determined by measuring the temperature dependence of the binding enthalpy (Eq 1).29 ∆ =

(∆ ) (1) ( ) 8

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These data for Co(II) binding to GpdQ in the presence of phosphate over the 15-30 °C temperature range (Figure S2) reveal a negative change in the heat capacity, estimated to be -260 and -205 cal/mol·K for the α and β sites, respectively. Negative ∆Cp° values for binding to proteins in aqueous solution indicate a decrease in the amount of ordered water, and have been associated with the sequestration of hydrophobic groups and/or release of solvent from the protein binding site.30-32 Finally, the affinities and thermodynamics of Co(II) binding to the Asn80Asp and Ser127Ala mutants of GpdQ were quantified by ITC, with representative data shown in Figure 4. For both proteins, two Co(II) ions now bind in the absence of phosphate, with a higher affinity for Co(II) at the α site (Table 2) than found with the wild-type protein. The number of protons displaced upon Co(II) binding to each site of these mutants was also determined (Figure S3). As found with wild-type GpdQ, both in the absence and presence of phosphate, approximately one proton is lost upon Co(II) binding to the α site but essentially none are lost upon binding to the β site. Analysis of these experimental data provides the condition-independent thermodynamics of Co(II) binding to the Asn80Asp and Ser127Ala mutants of GpdQ (Table 2).

Discussion Assembly of the catalytic active site of GpdQ has been investigated previously by structural, kinetic and spectroscopic techniques, using phosphate to mimic the binding interactions of substrates.15-18 In its resting state, the enzyme is predominantly mononuclear with a metal ion bound at the α site (Figure 1),15,18 which was confirmed and quantified here by ITC measurements of Mn(II) and Co(II) binding to apo GpdQ. The presence of substrate increases the affinity of the β site for metal ions, thereby forming the catalytically active binuclear enzyme. This was also confirmed and quantified here by ITC measurements of Mn(II) and Co(II) binding to GpdQ in the presence of phosphate. Two mutants of GpdQ, Asn80Asp and Ser127Ala, have been prepared to investigate perturbations of the first and second coordination sphere, respectively, in the β site. As shown by magnetic circular dichroism (MCD), both of these mutants have an increased affinity for Co(II) at the β site in the absence of phosphate.16,18 The Asn80Asp 9 ACS Paragon Plus Environment

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mutant has a carboxylate that coordinates metal ions more tightly than the native amide, and the Asn80 amide is a stronger Lewis base in the Ser127Ala mutant due to loss of the hydrogen bond from Ser127. As shown by activity measurements, coordination flexibility of the β site residue Asn80 (Figure 1) plays a crucial role in regulating GpdQ activity, with the Asn80Asp mutant, and its more strongly coordinating carboxylate, exhibiting considerably reduced activity.16,18 The Ser127Ala mutation, however, does not restrain the flexibility of Asn80, and this mutant retains high catalytic activity.19 Thus, this second sphere mutation increases the metal affinity of the β site in the resting state of the enzyme, yet does not significantly affect the substrate-bound state, resulting in an activity that is very similar to that of wild-type GpdQ.

α Site ITC measurements of wild-type GpdQ confirm that the α site binds Co(II) with higher affinity than it does Mn(II) (Kd1 = 0.7 and 5.4 µM, respectively), and reveal that the metal ion displaces one net proton at pH 7.25. While this could come from the two His residues in the α site (Figure 1), there is compelling evidence (vide infra) that it originates from a Co-bound water that is stabilized as hydroxide by hydrogen bonding in the site. The affinity of the α site for Co(II) and Mn(II) results from a favorable binding entropy (-T∆SCI = -10 and -15 kcal/mol, respectively) that overcomes an unfavorable binding enthalpy (∆HCI = +1.2 and +7.6 kcal/mol, respectively), which is more pronounced with Mn(II). Loss of the proton and desolvation of a well-ordered hydrophilic α site contribute to the entropically favored binding. The ITC data only allow an upper limit to be estimated for the metal affinity of the β site (Kd2 > 1 mM) in the wild-type protein. The affinity of the α site for Mn(II) determined by ITC is approximately five-fold higher than the value estimated previously by EPR spectroscopy, which also detected the weaker binding of a second Mn(II) in the absence of phosphate.16 These discrepancies relate to differences between the methods, data analysis and experimental conditions. EPR is very sensitive but only detects free Mn(II) in a titration with GpdQ. Further, Kd values for both sites were determined from the fit of a hyperbolic binding isotherm to a sequential binding model. ITC is unable to detect weak or iso-enthalpic (∆HITC ~ 0) 10 ACS Paragon Plus Environment

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binding, but it directly measures the heat flow associated with binding and can accurately quantify individual sequential binding events. Finally, EPR samples contained the tertiary amine buffer HEPES, while ITC samples contained the primary amine buffer Tris, and different interaction of these buffers with Mn(II) ions may contribute to the discrepancy. In the presence of phosphate, ITC measurements show that the α site has a ~75 fold higher affinity for Co(II) and again a single proton is displaced. This indicates that a Co(II)-bound hydroxide also forms when phosphate is bound. Phosphate increases the enthalpic binding penalty by +5 kcal/mol, but this is overcome by an even larger and favorable -7 kcal/mol entropic contribution to the binding. This suggests additional desolvation from the Co-bound phosphate in the protein site, consistent with the large negative ∆Cp° value for Co(II) binding with phosphate. A similar trend is found with Mn(II), which now binds with a ~35 fold higher affinity, although phosphate has a somewhat smaller effect on the Mn(II) binding thermodynamics. A higher affinity of the α site for Mn(II) in the presence of phosphate was found previously by EPR measurements, although a fit of the indirect binding data with this method indicated an increase of only a factor of two.16

β Site When there is a metal-bound phosphate in the α site of GpdQ, the β site has a significantly higher affinity for a second Co(II) or Mn(II) (Kd2 = 5 µM and 60 µM, respectively; Table 1). This value for Mn(II) is in good agreement with the value determined by EPR (Kd2 = 56 µM).16 In contrast to the α site, little if any proton displacement accompanies metal binding at the β site. Since both sites have two solventexposed metal-coordinating His residues (Figure 1), this discounts these residues as the source of the proton that is released upon metal binding at the α site. Binding at the β site is now favorable due to contributions of the phosphate (coordination, electrostatics), yet the Co(II) and Mn(II) binding thermodynamics are quite different. While Co(II) binding is enthalpically driven (∆HCI = -7 kcal/mol) with no net entropic contribution, Mn(II) binding is predominantly favored by entropic factors (-T∆SCI = -5 kcal/mol). Phosphate coordination to Co(II) in the β site has been demonstrated by MCD,15 and this contributes to the favorable binding enthalpy. The large negative value of ∆Cp° when Co(II) binds to 11 ACS Paragon Plus Environment

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the β site in the presence of phosphate indicates a significant loss of bound water. Therefore, the lack of an entropic contribution to binding suggests that desolvation of the β site is cancelled by conformational restrictions as the catalytic binuclear Co(II) site locks into place. Phosphate provides enthalpic contributions that are different for Co(II) and Mn(II) binding at the β site, reflecting differences in their metal-phosphate bond enthalpies. However, this substrate analog also modulates the entropic component of binding at this site, which is different for these two metal ions. Thus, the low affinity of the β site for metal ions in the absence of substrate is due to an insufficient enthalpic (bonding) contribution, as well as entropic factors, which are increased during substrate-promoted assembly of the binuclear GpdQ catalytic site.15,16

Mutations at the β Site The Asn80Asp and Ser127Ala mutants of GpdQ have a modified β site, as Asn80 is a metal-coordinating residue and Ser127 is hydrogen bonded to Asn80. Due to sequential metal binding, the effect of these mutations on the thermodynamics of Co(II) binding to each site can be evaluated independently for insight into the formation of the binuclear catalytic site. Both of these mutations increase the affinity of the α site for Co(II) to a similar extent, relative to wild-type GpdQ. This results from a more favorable binding enthalpy (∆∆HCI ~ -13 kcal/mol), yet a significant, though smaller, less favorable binding entropy (-T∆∆SCI ~ +12 kcal/mol). Thus, replacing Asn80 with a negatively charged Asp or eliminating the hydrogen bond from Ser127 to the metal-coordinating amide of Asn80 in the β site has a significant and similar effect on both the enthalpic and entropic components of metal binding at the α site. These distal and opposing thermodynamic contributions reflect an enthalpy-entropy compensation (EEC) within an extensively hydrogen bonded enzyme active site. Both of these mutations increase the affinity of the β site for Co(II) to a similar value in the absence of phosphate. Thus, replacing an Asn with an Asp or eliminating the hydrogen bond to Asn80 results in the same overall effect on metal binding at the β site. In both cases this affinity is due to a favorable binding enthalpy that overcomes an 12 ACS Paragon Plus Environment

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unfavorable binding entropy. The enthalpic component can be attributed to stronger bonding of the carboxylate of the Asn80Asp mutant and the unconstrained Asn80 amide of the Ser127Ala mutant. It is important to note, however, that the magnitude of these opposing thermodynamics is remarkably large in the case of the Ser127Ala mutant (∆HCI = -37 ± 5 kcal/mol; -T∆SCI = +31 ± 2 kcal/mol). Clearly, this second-sphere hydrogen bond suppresses Asn80 bonding to the metal, yet its loss by replacing the polar Ser with a hydrophobic Ala also has significant conformational and/or solvation effects at the β site. Co(II) binding to both mutants in the presence of phosphate provides further insight. This substrate analog has no additional effect on the Co(II) affinity and binding thermodynamics of the α site of these mutants. However, the effect of phosphate at the β site allows two comparisons to be made, one with the wild-type protein in the presence of phosphate and the other with these mutants in the absence of phosphate. The former reveals the effect of the mutation on the formation of the catalytic binuclear site, while the latter indicates the contribution of the substrate analog to metal binding. Comparison of the wild-type and mutant proteins in the presence of phosphate reveals that both mutations lower the Co(II) affinity of the β site due to a proportionally larger entropic penalty. Thus, while these mutations increase the metal affinity of the β site in the absence of phosphate, they cancel some of the phosphate contributions to metal binding at this site. It is important to note that again the Ser127Ala mutant has a much more favorable binding enthalpy (∆∆HCI = -18 kcal/mol) that is cancelled by a larger unfavorable binding entropy (-T∆∆SCI = +19 kcal/mol). This mutation, which eliminates a hydrogen bond by substituting a hydrophobic residue (Ala) for a polar residue (Ser), clearly introduces a significant perturbation of the active site coordination, solvation, hydrogen bonding and/or dielectric. Comparison of the mutants with and without phosphate reveals that this substrate analog has a negligible effect on the Co(II) affinity of the β site of both mutants. This results from an unfavorable contribution to the binding enthalpy for both mutants (∆∆HCI = +5 and +12 kcal/mol, respectively) that is cancelled by a favorable contribution to their binding entropy (-T∆∆SCI = -6 and -12 kcal/mol, respectively). In this case of EEC, favorable conformational or solvation effects of the phosphate cancel an unfavorable contribution to the metal binding enthalpy that it imposes at the mutant β sites. Once 13 ACS Paragon Plus Environment

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again, a larger effect on the binding thermodynamics is found with the Ser127Ala mutant, further indicating the importance of this residue and its hydrogen bond to the metalcoordinating Asn80 residue.

Formation of the GpdQ Catalytic Site The generally accepted mechanism for substrate hydrolysis by GpdQ, and other binuclear metallohydrolases, includes a metal-activated solvent nucleophile, and structural15 and electronic18 data with excess metal ions indicate that a hydroxide bridges the two metal ions. We have shown here with ITC measurements that only a single proton is released during assembly of the binuclear metal site and that this deprotonation accompanies the first metal binding. While the second metal binding to the native protein requires the substrate analog phosphate, the Ser127Ala mutant binds both metal ions in the absence of phosphate and has high activity, indicating that the catalytic hydroxide nucleophile of GpdQ is created when a metal ion binds at the α site. Therefore, the inactive mononuclear resting form of GpdQ has a metal-bound hydroxide at pH 7.25, but is not competent to catalyze the hydrolysis of substrates until a second metal ion binds at the β site, which may further activate and/or position the nucleophile, as well as the substrate. In a recent study of the metallo-β-lactamase from Bacillus cereus, BcII, Page and co-workers used ITC to measure Zn(II), Co(II) and Cd(II) binding to the protein over the pH range 5.2-7.2 with different buffers, allowing proton displacement to be determined.33 At pH > 6.0 the protein binds two metal ions at an asymmetric active site with His3 and AspCysHis residue coordination and a bridging hydroxide. Experimental binding constants for both metal ions were extracted from the binding isotherms, although the individual metal-binding thermodynamics were not determined. Since the two binding events for Zn(II) and Co(II) are not resolved, as they are for GpdQ with phosphate and the two GpdQ variants, proton displacement could only be reported for complete formation of the binuclear active site with its bridging hydroxide. Therefore, insight about the stepwise assembly of the binuclear catalytic site of GpdQ and formation of the nucleophilic hydroxide, which we report here, is not available for BcII.


Page 14 of 24

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Biochemistry

Finally, comparisons of the metal-binding thermodynamics of wild-type GpdQ and the two mutant proteins in the absence and presence of phosphate reveal cases of enthalpy-entropy compensation, which are pronounced with the second-sphere mutant Ser127Ala, that result from extensive hydrogen bonding in the GpdQ active site. There is special interest in this mutant for use in bioremediation, since it has both high activity and a high affinity for both metal ions, thereby eliminating the need for activation with metals prior to its application.20,34

Acknowledgements: GS acknowledges the receipt of an ARC Future Fellowship (FT120100694).

Supporting Information Available: plots of experimental enthalpies vs buffer protonation enthalpies to determine proton displacement upon metal binding; temperature-dependence of the binding enthalpy to determine ∆Cp°.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References 1. Schenk, G., Mitić, N., Gahan, L. R., Ollis, D. L., McGeary, R. P. and Guddat, L. W. (2012) Binuclear metallohydrolases: complex mechanistic strategies ofr a simple chemical reaction, Acc. Chem. Res. 45, 1593-1603. 2. Zambelli, B., Muiani, F., Benini, S. and Ciurli, S. (2011) Chemistry of Ni2+ in urease: sensing, trafficking and catalysis, Acc. Chem. Res. 44, 520-530. 3. O’Brien, P. J. and Herschlag, D. (2001) Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of E. coli alkaline phosphatase, Biochemistry 40, 5691-5699. 4. Cama, E., Pethe, S., Boucher, J.-L., Han, S., Emig, F. A., Ash, D. E., Viola, R. E., Mansuy, D. and Christianson, D. W. (2004) Inhibitor coordination interactions in the binuclear manganese cluster of arginase, Biochemistry 43, 8987-8999. 5. D’souza, V. M., Bennett, B., Copik, A. J. and Holz, R. C. (2000) Divalent metal binding properties of the methionyl aminopeptidase from E. coli, Biochemisty 40, 56915699. 6. Llarrull, L. I., Tioni, M. F. and Vila, A. J. (2008) Metal content and localization during turnover in B. cerus metallo-β-lactamase, J. Am. Chem. Soc. 130 15842-15851. 7. Wilcox, D. E. (1996) Binuclear metallohydrolases, Chem. Rev. 96, 2435-2458. 8. Meini, M.-R., Llarrull, L. I. and Vila, A. J. (2015) Overcoming differences: the catalytic mechanism of metallo-β-lactamases, FEBS Lett. 589, 3419-3432. 9. Mitić, N., Smith, S. J., Neves, A., Guddat, L. W., Gahan, L. R. and Schenk, G. (2006) The catalytic mechanisms of binuclear metallohydrolases, Chem. Rev. 106, 3338-3363. 10. Jackson, C. J., Carr, P. D., Liu, J.-W., Watt, S. J., Beck, J. L. and Ollis, D. L. (2007) The structure and function of a novel glycerophosphodiesterase from Enterobacter aerogenes, J. Mol. Biol. 367, 1047-1062. 11. Schenk, G., Mitić, N., Hanson, G. R. and Comba, P. (2013) Purple acid phosphatase: a journey into the function and mechanism of a colorful enzyme, Coord. Chem. Rev. 257, 473-482. 12. Shenoy, A. R., Capuder, M., Draskovic, P., Lamba, D., Visweswariah, S. S. and Podobnik, M. (2007) Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis, J. Mol. Biol. 365, 211-225. 13. Keppetipola, N. and Shuman, S. (2008) A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity, J. Biol. Chem. 283, 30942-30949. 14. Ghanem, E., Li, Y., Xu, C. and Raushel, F. M. (2007) Characterization of a phosphodiesterase capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve agent VX, Biochemistry 46, 9032-9040. 15. Hadler, K. S., Tanifum, E. A., Yip, S. H.-C., Mitic, N., Guddat, L. W., Jackson, C. J., Gahan, L. R., Nguyen, K., Carr, P. D., Ollis, D. L., Hengge, A. C., Larrabee, J. A. and Schenk, G. (2008) Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes, J. Am. Chem. Soc. 130, 14129-14138. 16. Hadler, K. S., Mitic, N., Ely, F., Hanson, G. R., Gahan, L. R., Larrabee, J. A., Ollis, D. L. and Schenk, G. (2009) Structural flexibility enhances the reactivity of the bioremediator glycerophosphodiesterase by fine-tuning its mechanism of hydrolysis, J. Am. Chem. Soc. 131, 11900-11908. 16 ACS Paragon Plus Environment

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Biochemistry

17. Hadler, K. S., Gahan, L. R., Ollis, D. L. and Schenk, G. (2010) The bioremediator glycerophosphodiesterase employs a non-processive mechanism for hydrolysis, J. Inorg. Biochem. 104, 211-213. 18. Hadler, K. S., Mitic, N., Yip, S. H.-C., Gahan, L. R., Ollis, D. L., Schenk, G. and Larrabee, J. A. (2010) Electronic structure analysis of the dinuclear metal center in the bioremediator glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes, Inorg. Chem. 49, 2727-2734. 19. Daumann, L. J., McCarthy, B. Y., Hadler, K. S., Murray, T. P., Gahan, L. R., Larrabee, J. A., Ollis, D. L. and Schenk, G. (2013) Promiscuity comes at a price: catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ, Biochim. Biophys. Acta 1834, 425-432. 20. Daumann, L. J., Larrabee, J. A., Ollis, D., Schenk, G. and Gahan, L. R. (2014) Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation, J. Inorg. Biochem. 131, 1-7. 21. Pedroso, M. M., Ely, F., Lonhienne, T., Gahan, L. R., Ollis, D. L., Guddat, L. W. and Schenk, G. (2014) Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry, J. Biol. Inorg. Chem. 19, 389-398. 22. MicroCal. (2004) ITC Data analysis in Origin - tutorial guide, MicroCalorimeter User`s Manual - Northampton, MA. 23. Lin, L. N., Mason, A. B., Woodworth, R. C. and Brandts, J. F. (1991) Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites, Biochemistry 30, 11660-11669. 24. Lin, L. N., Mason, A. B., Woodworth, R. C. and Brandts, J. F. (1993) Calorimetric studies of the binding of ferric ions to human serum transferrin, Biochemistry 32, 93989406. 25. Grossoehme, N. E., Akilesh, S., Guerinot, M. L. and Wilcox, D. E. (2006) Metalbinding thermodynamics of the histidine-rich sequence from the metal-transport protein IRT1 of Arabidopsis thaliana, Inorg. Chem. 45, 8500-8508. 26. Grossoehme, N. E., Spuches, A. M. and Wilcox, D. E. (2010) Application of isothermal titration calorimetry in bioinorganic chemistry, J. Biol. Inorg. Chem. 15, 1183-1191. 27. Rich, A. M., Bombarda, E., Schenk, A. D., Lee, P. E., Cox, E. H., Spuches, A. M., Hudson, L. D., Kieffer, B. and Wilcox, D. E. (2012) Thermodynamics of Zn(II) binding to Cys2His2 and Cys2HisCys zinc fingers and a Cys4 transcription factor site, J. Am. Chem. Soc. 134, 10405-10418. 28. Quinn, C. F., Carpenter, M. C., Croteau, M. L. and Wilcox, D. E. (2016) Isothermal titration calorimetry of metal ions binding to proteins, Meth. Enzymol., Vol. 567, A. Feig, Ed., Burlington: Academic, 3-21. 29. Thomson, J., Ratnaparkhi, G. S., Varadarajan, R., Sturtevant, J. M. and Richards, F. M. (1994) Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S, Biochemistry 33, 8587-8593. 30. Sturtevant, J. M. (1977) Heat capacity and entropy changes in processes involving proteins, Proc. Nat. Acad. Sci. U.S.A. 74, 2236-2240. 31. Ross, P. D., Hofrichter, J. and Eaton, W. A. (1975) Calorimetric and optical characterization of sickle cell hemoglobin gelation, J. Mol. Biol. 96, 239-253.


Biochemistry

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32. Brandts, J. F. (1964) The thermodynamics of protein denaturation. II. a model of reversible denaturation and interpretations regarding the stability of chymotrypsinogen, J. Am. Chem Soc. 86, 4302-4314. 33. Motara, H., Mistry, D., Brown, D. R., Cryan, R. A., Nigen, M. and Page, M. I. (2014) pH and basicity of lignds control the binding of metal ions to B. cereus B1 β-lactamase, Chem. Sci. 5, 3120-3129. 34. Yip, S. H., Foo, J. L., Schenk, G., Gahan, L. R., Carr, P. D. and Ollis, D. L. (2011) Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme, Prot. Eng. Des. Sel. 24, 861-872.


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Biochemistry

Table 1. Average best-fit experimental ITC values and condition-independent thermodynamic values for Mn(II) and Co(II) binding to GpdQ at pH 7.25 and 25 °C. wt-GpdQa,b

wt-GpdQ + Phosphatea,c

α- metal site

β- metal site

α- metal site

β- metal site

1.0(0.1)

-

0.98(0.07)

1.2(0.1)

-4.6(0.2)

-

-0.90(0.05)

-2.9(0.7)

KITC

1.6(0.1) x 105

-

5.3(0.7) x 106

6.1(0.2) x 104

ΔG°CI d,e

-7.2(0.3)

-

-9(1)

-5.8(0.3)

ΔHCI d,e,f

7.62(0.05)

-

11.3(0.3)

-0.9(0.5)

-TΔSCI d,e

-14.8(0.1)

-

-20.5(0.7)

-4.8(0.2)

Mn(II) nITC ΔHITC

d

1.8(0.1) x

K Kd (µM)

105

-

5.4(0.3)

-

6.2(0.1) x

106

1.7(0.2) x 104

0.16(0.03)

60.2(0.3)

Co(II) nITC

1.0(0.2)

1.3(0.2)

1.2(0.2)

ΔHITC d

-8.3(0.7)

-3(1)

-6(1)

KITC

1.0(0.1) x 106

7.1(0.3) x 107

3.1(0.6) x 105

ΔG°CI d,e

-8.45(0.03)

-

-11(2)

-7(1)

ΔHCI d,e,f

1.2(0.3)

-

6(2)

-7(1)

-9.7(0.9)

-

- 17(1)

-0.1(0.2)

K

1.6(0.4) x 106

-

1.1(0.9) x 108

2.1(0.3) x 105

Kd (µM)

0.67(0.01)

-

0.0090(0.0007)

4.7(0.1)

-TΔSCI

a

d,e

.

All measurements were obtained in 50 mM Tris HCl buffer, pH 7.25. Fitted with a one-site binding model. c Fitted with a two independent sites binding model. d kcal/mol e Condition independent (CI) values at pH 7.25. f Values were calculated using the equation: ∆ = ∆ + ∆ − ∆ !. b


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

Table 2. Average best-fit experimental ITC values and condition-independent thermodynamic values for Co(II) binding to the GpdQ mutants Ser127Ala and Asn80Asp at pH 7.25 and 25 °C. Ser127Ala a

Ser127Ala + Phosphatea

α- metal site

β- metal site

α- metal site

β- metal site

1.3(0.3)

0.8(0.6)

1.1(0.3)

0.9(0.4)

-21(3)

-36(3)

-18(2)

-24(3)

KITC

9.2(0.1) x 106

2.2(0.2) x 104

9.6(0.1) x 106

4.2(0.1) x 104

ΔG°CI b,c

-10(1)

-5.7(0.8)

-10(2)

-6(2)

ΔHCI b,c,d

-11(2)

-37(5)

-9(2)

-25(1)

-TΔSCI b,c

1(1)

31(2)

-1(3)

19(2)

K

1.5(0.1) x 107

1.5(0.5) x 104

1.5(0.3) x 107

2.9(0.1) x 104

Kd (µM)

0.07(0.07)

67(7)

0.06(0.02)

34(3)

nITC ΔHITC

b

Asn80Asp a

a

Asn80Asp + Phosphatea

α- metal site

β- metal site

α- metal site

β- metal site

nITC

0.90(0.06)

1.1(0.1)

0.91(0.06)

1.0(0.8)

ΔHITC b

-22(3)

-12(1)

-21.2(0.9)

-7(1)

KITC

6.2(0.2) x 106

1.2(0.1) x 104

8.5(0.8) x 106

2.9(0.2) x 104

ΔG°CI b,c

-10(2)

-5.3(0.9)

-10(2)

-6(1)

ΔHCI b,c,d

-12(2)

-13(3)

-12(3)

-8(1)

-TΔSCI b,c

3(1)

7.8(0.8)

2(2)

2(3)

K

9.8(0.1) x 106

8.2(0.6) x 103

1.3(0.5) x 107

2.0(0.7) x 104

Kd (µM)

0.10(0.09)

120(9)

0.07(0.08)

51(8)

.

All measurements were obtained in 50 mM Tris HCl buffer, pH 7.25; data were fitted with a two independent sites binding model. b kcal/mol c Condition independent (CI) values at pH 7.25. d Values were calculated using the equation: ∆ = ∆ + ∆ − ∆ !


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure Captions Figure 1. Illustrative structures of the active sites of wild-type GpdQ and its Asn80Asp and Ser127Ala mutant forms. Figure 2. Representative ITC data (600 sec between injections) for the addition of MnCl2 (2 mM) into GpdQ (150 µM), left, and CoSO4 (2 mM) into GpdQ (170 µM), right, in 50 mM Tris, pH 7.25; the data were fitted using a one-site binding model. Figure 3. Representative ITC data for the addition of MnCl2 (2 mM; 600 sec between injections) to GpdQ (150 µM), left, and CoSO4 (2 mM; 400 sec between injections) to GpdQ (140 µM), right, in 50 mM Tris, pH 7.25, with 1.5 mM K2HPO4; the data were fitted using a two independent sites binding model, with the early red points in the MnCl2 titration masked for the fit. Figure 4. Representative ITC data (150 sec between injections) for the addition of CoSO4 (2 mM) to the GpdQ mutants Ser127Ala (120 µM), left, and Asn80Asp (150 µM), right, in 50 mM Tris, pH 7.25; the data were fitted using a two independent sites binding model.


Biochemistry

Asp50

Asp50 O

Asp8 His10

H N

O O

O

His 10

N

α

N HN

H 2O

N H

β H

His197

Ser127

Wild-Type

N

Asp80

O

O

H

H 2O

N H

NH

His156

β

N

H O

N

O

α

HN H 2N

H N

O

O N

O

O

N His197

O

Asp8

His156

Asn80

OH

His195

Asn80Asp

Asp50 O

His10

H N

O O

O

His156

N Asn80

N

α

HN

N H

O NH 2

O

N His 197

β

H 2O

H

N Ala127 NH

Ser127Ala

His195

Figure 1

Time (min)

Time (min) 0

50 100 150 200 250 300 350 400

0.00

0.00

-0.50

-0.50

µcal/sec

µcal/sec

0

-1.00

50 100 150 200 250 300 350 400

-1.00

-1.50

-1.50

0.00 0.00

kcal/mol

-2.00 -2.00

-4.00

-4.00 -6.00 -8.00

0

2

4

6

8

0

10

Molar Ratio

1

2

3

4

Molar Ratio

Figure 2


O-

N NH

His195

Asp8

kcal/mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

5

6

Ser127

Page 23 of 24

Time (min)

Time (min)

0

-100 0 100 200 300 400 500 600 700 800

100

200

300

400

500

0.00

0.00

-0.10

µcal/sec

µcal/sec

-0.10

-0.20

-0.20 -0.30

-0.30

-0.50

0.00

0.00

kcal/Mole of Injectant

kcal/Mole of Injectant

-0.40

-0.50 -1.00 -1.50

-1.20 -2.40 -3.60 -4.80

-2.00

-6.00 0

1

2

3

4

0

5

1

2

3

4

5

6

Molar Ratio

Molar Ratio

Figure 3 Time (min)

Time (min) 0

50

100

150

200

0

33

0

1

67

100

133

167

200

0.00

0.00

-1.00

µcal/sec

µcal/sec

-1.00 -2.00 -3.00

-2.00 -3.00 -4.00

-4.00

-5.00 -5.00 0.00

0.00

-5.00

-5.00 kcal/mol

kcal/mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

-10.00 -15.00

-10.00 -15.00 -20.00

-20.00

-25.00

-25.00 0

1

2

3

4

5

2

3

Molar Ratio

Molar Ratio

Figure 4


4

5

7

8

Biochemistry

For Table of Contents Use Only (Table of Contents Graphic)

Asp50

0.00 kcal/Mole of Injectant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

O

Asp8 His10

-1.20

H N

O O

O

His156

N Asn80

α

N

-2.40

HN N

-3.60

His197

N H

β

O H 2N

O H 2O

H

N NH

-4.80 Wild-Type

-6.00 0

1

2

3

4

5

6

7

8

Molar Ratio


His195

H O

Ser127

Mechanistic Insight from Calorimetric Measurements of the Assembly

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