Substitution of Calcium with Divalent Metal Ions in Paraoxonase I

MASTER THESIS

Presented in Partial Fulﬁllment of the Requirements for the Degree Master Degree in the Graduate School of The Ohio State University

By Yu-Wen Wang, M.S. Graduate Program in Chemistry and Biochemistry

The Ohio State University 2015

Master Thesis Committee: Terry Gustafson, Advisor

Tom Magliery, Advisor c Copyright by

Yu-Wen Wang

2015 Abstract

In order to understand the hydrolysis mechanism of Paraoxonase I (PON1), the catalytic calcium in PON1 has been substituted with other cations. In each substitution, the catalytic calcium was ﬁrst removed by equilibrium dialysis of metal chelating agents EDTA, and then the investigated cation was added into the dialyzed solution. Experimental results show that the activity of PON1 is regained and activities are varied with diﬀerent cations. The data are interpreted in the context of tendency of pKa values of cations, stability constants, dissociative, and associative mechanisms preferences of investigated cations, and the solvent-metal exchange rates. The results suggest that dissociative mechanism is relative preferred to be the mechanism of PON1.

ii Table of Contents

Page Abstract...... ii List of Figures ...... v List of Tables ...... vii

Chapters

1 Introduction1 1.1 Paraoxonase-I...... 1 1.2 Nucleophilic Attack Six-Bladed Beta-Propeller (N6P) Superfamily..... 4 1.2.1 SMP-30...... 5 1.2.2 Diisopropyl ﬂuorophosphatase (DFPase)...... 7 1.3 Promiscuity of Organophosphatases ...... 8 1.4 Metal Substitution...... 9 1.4.1 Case Study: Phosphortriesterase...... 10 1.4.2 Other Methods for Metalloprotein Analysis...... 15 1.5 Previous Metal Substitution in PON1 ...... 17

2 Methods 20 2.1 Expression of Paraoxonase I...... 20 2.2 Bradford Assay...... 21 2.3 Apo-Paraoxonase I - Dialysis Processes...... 21 2.4 Kinetic Measurement...... 22 2.4.1 5-thiobutyl butyrolactone(TBBL) Measurement...... 23 2.4.2 Paraoxon and Dihydrocoumarin Measurement...... 24 2.5 Calculation ...... 24

3 Results 26 3.1 EDTA treatment of Paraoxonase I ...... 26 3.2 Substitution Other Divalent/Trivalent Metal Ion for Calcium Ion ...... 29 3.2.1 pH value Factor ...... 30 3.2.2 Metal Factor ...... 30 3.3 Negative Control - Hydrolysis Rate of Pure Metal Solution ...... 31 3.4 Negative Control - Bovine Serum Albumin(BSA) Measurement ...... 32

4 Discussion 33

iii 4.1 Metal-Ligands/Water Molecules Aﬃnity...... 33 4.2 Transition Metal - Irving-Williams series (pH7.5)...... 36 4.3 Dissociative and Associative Mechanisms ...... 36 4.4 Transition Metal - Ligand Exchange Rate (pH9.0) ...... 37 4.5 Metal-Substrates Aﬃnity ...... 38

5 Conclusion 40

Appendices

A pH Dependent PON1 Activity 50

B Jahn-Teller Distortion and Lability of Cu(II) 58

iv List of Figures

Figure Page

1.1 Crystal structure of RePON1...... 2 1.2 The suggested mechanisms of PON1...... 3 1.3 Structural homologues of PON1 in N6P superfamily...... 4 1.4 Structure and active site of SMP-30 ...... 6 1.6 Nuclear density map of DFPase active site...... 7 1.5 Schematic representation of possible mechanisms for DFPase...... 8 1.7 Simpliﬁed energy diagram of promiscuous enzyme and the cartoon of pocket9 1.8 Schematic representation of the bi-metallic centre of OPHC2 ...... 11 1.9 The suggested mechanism for phosphortriesterase...... 12 1.10 The schematic concept of MCD...... 12 1.11 Summary of ligand ﬁeld calculations in MCD spectra...... 13 1.12 Energy diagram of the lanthanide complex, consisting of a sensitizer and a lanthanide(III) ion. A good sensitizer such as tryptophan (Trp) ...... 16 1.13 Images of Zn, Cu, Fe, Pb, Mn and Ag in 2D-blue native gel electrophoresis gel of rat kidney water extract ...... 16 1.14 Fluorescence spectrum of Tb3+-replaced PON1...... 17 1.15 Inhibition patterns of additional Mg2+, Sr2+, Cu2+ and Ba2+ ...... 18

2.1 PON1 G3C9 Gel...... 20 2.2 Mechanism of DNTB as indicator of TBBL hydrolyzation...... 23 2.3 Calculation process...... 24

3.1 Dialysis eﬃciency...... 26 3.2 Concentration of rebound cation ...... 27 3.3 Stability of metal-substituted PON1s: Px...... 28 3.4 Stability of metal-substituted PON1s: DHC...... 28 3.5 Activity of metal substitution at pH 7.5...... 29 3.6 Activity of metal substitution at pH 9.0...... 30 3.7 Negative control - Hydrolysis rate of Px with additional cation solutions . . 31 3.8 Negative control - Hydrolysis rate of DHC with additional cation solutions . 31 3.9 Negative control - Hydrolysis rate of Px by BSA with additional cation solutions 32

v 3.10 Negative control - Hydrolysis rate of DHC by BSA with additional cation solutions...... 32

4.1 Swaddles scaled More-OFerrall diagram assuming a continuum of interchange mechanisms...... 37 4.2 Mean lifetimes of a particular water molecule in the ﬁrst coordination shell of a given metal ion and the corresponding water exchange rate ...... 38

5.1 Solvent exchange processes: volume proﬁles connected to the transition states 40

A.1 pH value dependent PON1 activity - Px...... 54 A.2 pH value dependent PON1 activity - TBBL...... 55 A.3 pH value dependent PON1 activity - PA...... 56 A.4 pH value dependent PON1 activity - DHC...... 57

vi List of Tables

Table Page

1.1 Experimentally characterized activities of selected N6P superfamily enzymes5 1.2 Kinetic characterization of SMP-30 with gluconolactone and its metal dependence ...... 7

2.1 Substrate list...... 22

4.1 Experimentally determined pKa value and calculated deprotonation energies for the d-block metal complexes...... 34 n+ 4.2 Standard Reduction Potentials of M(OH)n and M ...... 34 4.3 Stability Constants (log K1) of Various Metal Chelates...... 35

A.1 Specific Activities of metal-substituted PON1s at pH 6.0, unit: mM−1 min−1 50 A.2 Specific Activities of metal-substituted PON1s at pH 6.5, unit: mM−1 min−1 51 A.3 Specific Activities of metal-substituted PON1s at pH 7.0, unit: mM−1 min−1 51 A.4 Specific Activities of metal-substituted PON1s at pH 7.5, unit: mM−1 min−1 52 A.5 Specific Activities of metal-substituted PON1s at pH 8.0, unit: mM−1 min−1 52 A.6 Specific Activities of metal-substituted PON1s at pH 8.5, unit: mM−1 min−1 53 A.7 Specific Activities of metal-substituted PON1s at pH 9.0, unit: mM−1 min−1 53

vii Chapter 1 Introduction

1.1 Paraoxonase-I

Organophosphates (OPs) includes insecticides, chemical weapons and nerve agents, are well-known for their threat to human health. The damage is caused by inhibition of OPs to acetylcholinesterase (AChE), which is a hydrolase in the synaptic cleft and controls the level of neurotransmitter acetylcholine (ACh). Since the AChE is inhibited by OPs, it would raise the concentration of ACh, overstimulate the neuron receptors, and overproduce nerve impulses. The abnormally high intensity of nerve impulses results in miosis, bradycardia, fasciculation and convulsions leading to death due to respiratory failure. One of the treatments for nerve agent exposure is to reduce the OPs level in the human body by bioscavengers. The chosen/modified bioscavengers should have the following properties: (1) catalytic efficiency; (2) immunotolerance; (3) resistant to rapid degradation within the body; and (4) no adverse side effects [1]. Some of potential candidates which fit those requirement are Butyrylcholinesterase (BuChE), Phosphotriesterase(PTE), and paraoxonase 1 (PON1). PON1 (Paraoxonase-1, E.C.3.1.1.2) [2–6] is a mammalian serin protein which can be arylesterase and organophosphatase. As an organophosphatase, PON1 can hydrolyze parathion, chlorpyrifos, diazinon, sarin, and soman nerve agents [7]. Even though PON1 shows activity to OPs and does not have immunotolerance and toxicity issues, the overall efficiency is unsatisfied. Hense, for enhancement of the catalytic efficiency, scientists try to comprehensively investigate the structural properties and mechanism of PON1. PON1 is a membrane protein which has 335 amino acids (MW ∼ 44kD). The known structure of PON1 is recombinant PON1-G2E6 (rePON1), which has ∼ 90% identical se- quence to both rabbit and human PON1s and displays the same enzymatic specificity [8]. As shown in fig 1.1, PON1 has a six-bladed β-propeller with three α-helixes on the top and two calcium binding sites. Previous research suggested that two individual calcium ions actually play different roles in PON1. The calcium ion in the center of the tunnel (black

1 sphere) is considered to be the structural calcium, which is relatively buried and believed to stabilize the structure. In contrast, the calcium that is located close to the top of the surface is called catalytic calcium (gray sphere), and believed to be involved in the hydrolyzation. Besides, the binding aﬃnity of the structural calcium is found to be two order greater than the catalytic calcium [9]. This result supports the assumption since removing the structural metal would unfold PON1, the binding aﬃnity must be higher.

Figure 1.1: Crystal structure of RePON1 [8]. PON1 has a six-bladed β-propller structure with two calcium inside (black sphere and gray sphere). The calcium(black) in the center of PON1 is structural calcium and the one(gray) close to the surface of PON1 is catalytic calcium. Three α-helixes are on the top of the PON1: residues 1-15 at the N-termal(blue, H1), residues 189-198(green, H2) and residues 288-293(yellow, H3). As a membrane protein, H1 inserts into HDL and H2 contacts with the surface of HDL.

2 Figure 1.2: The suggested mechanisms of PON1 [10]. Tawﬁk et al. postulated that PON1 undergoes the general base catalysis, which the water molecule is activated by H115 or D269 attacks substrate. Whereas Worek et al. assumed that PON1 has direct hydrolysis by a nucleophilic residue D269.

PON1 is a promiscuous enzyme, which means it can hydrolyze more than one kind of substrate (the definition of promiscuity of enzyme is in sec 1.3). It can function as lactonase and organophosphatase. To date, the mechanism of PON1 is still under research, and two hypotheses of mechanisms have been suggested by Tawfik and Worek [10]. Based on the promiscuity property of PON1, Tawfik proposed PON1 has different mechanisms for lactones and organophosphates. In other word, Tawfik assumes PON1 is supposed to be a catalytic promiscuous enzyme, which hydrolyzes different kinds of substrates with different mechanisms. These two mechanisms by Tawfik are shown in fig 1.2. For lactonase activity, Tawfik suggests that the water molecule is activated by H115/H134 and E53, directly attacks lactones; for organophosphatase activity, the water is orientated and activated by E53 and D269, then the activated water directly attacks organophosphates. In contrast, Worek

3 et al postulated that PON1 hydrolyzes different kinds of substrates with one mechanism. Lactones and organophosphates are all hydrolyzed directly by D269. However, researches [11, 12] have proved that D269 mutant still has ∼1% activity, and the intermediate did not generate the PON1-substrate complex. The last one in the fig 1.2 is the alternative mechanism based on Worek’s postulation. The water molecule which bound to the catalytic calcium would be triggered by D269, then the activated water would attack phosphorous center and hydrolyze substrates. Moreover, another missing piece in this mechanism puzzle is the role of the catalytic calcium ion. PON1 has proved to be a calcium dependent protein [11, 13]. In order to understand the role of metal in PON1, several groups have done relative experiments. Pla and his group [13] used Mn2+, Co2+, Zn2+, Ba2+, Cu2+, La3+, Hg2+ and Mg2+ to inhibit microsomal PON1 in rat plasma and liver. As results, they found the order of inhibiting potency was Hg2+ > Co2+ > Mn2+ > Cu2+ for PON1. Also, Ben-David and his group [11] found that when another cation solution(Mg2+, Sr2+, Cu2+and Ba2+) is added into PON1 solution, the activity of PON1 to paraoxon will be inhibited. These experiments showed that how additional divalent cations in the solution affect the activity of PON1. However, the inhibition may due to the change of the solutions ionic strength instead of metal substitution. And it is still unclear about the exact role of catalytic calcium in the hydrolyzation. In our experiment, we substituted other metals for calcium in PON1. We used ethylene- diaminetetraacetic acid (EDTA) to produce apo-PON1 and filled in the active site with other metal cations of interest. After the substitutions, we measured the activities of the substituted PON1 with different kinds of substrates for further understanding of calcium in PON1.

1.2 Nucleophilic Attack Six-Bladed Beta-Propeller (N6P) Su- perfamily

Figure 1.3: Structural homologues of PON1 in N6P superfamily: The top-view of three six-bladed β-propeller folds. SMP-30 (PDB code 3g4e); Paraoxonase 1 (PDB code 1v04); DFPase (PDB code 1pjx) [14]. 4 PON1 belongs to nucleophilic attack six-bladed β-propeller (N6P) superfamily [15], which has following characteristics:

1. Compared to any other protein with similar folding, proteins in N6P superfamily share highly similar sequences with each other.

2. Members in the N6P superfamily mostly have the same catalytic characteristics: nucleophilic attack on an electrophilic substrate.

3. Almost all members in the N6P superfamily are metal dependent. Within these metal-dependent enzymes, the metal binding coordination and relative ligands are high conserved. Furthermore, N6P has an unique the fold class.

Table 1.1: Experimentally characterized activities of selected N6P superfamily enzymes [14]

Proteins Subgroup Lactonase Esterase Organophosphatase PON1 Arylesterase-like Yes Yes Yes SMP-30 SGL Yes Yes Yes DFPase SGL No ? Yes

N6P superfamily has three main subgroups: (1) The senescence marker protein-30/ gluconolactonase/ luciferin-regenerating enzyme-like (SGL) subgroup; (2) The strictosidine synthase-like (SSL) subgroup; and (3) The arylesterase-like subgroup. PON1 is belongs to the arylesterase-like subgroup. Within the members in N6P superfamily, the SGL subgroup has some proteins that are known to have the same/similar folding structure (shown in ﬁg 1.3) and substrates as PON1, such as senescence marker protein-30 (SMP-30) and di- isopropylphosphoﬂuoridate hydrolase (DFPase). Hence, previous research of those enzymes (table 1.1), may provide some hints for understanding PON1.

1.2.1 SMP-30

SMP-30 [14, 16, 17] is a six-bladed beta propeller has only one divalent cation binding site, as shown in fig 1.4(a)(b). The native function of SMP-30 is a lactonase, but it can hydrolyze organophosphates such as sarin, soman and diisopropylphosphofluoridate (DFP), when magnesium is bound to the active site. From the coordination of the SMP-30 active site, the divalent cation is bound with E18, N154, and D204 (fig 1.4(c)(d)). N103 and D104 are near the Ca2+, but not within bonding distance. The hydrolysis mechanism of SMP-30 is still unclear. Chakraborti et al. [16] demonstrate that E18, N103, N154 and D204 significantly affect the specific activity, especially D204. 5 Figure 1.4: (a) Top view of the crystal structure of human SMP-30 with Ca2+ bound. (b) Side view of SMP-30. (c) Electron density map and (d) scheme of the human SMP-30 metal binding site, around highlighted residues and three water molecules. [16].

However, Belinskaya et al. [17] pointed out that the D204 cannot become a nucleophile since there is no proton acceptor next to D204, which was also proved by Chakraborti’s experimental results. D204, the mutant of SMP-30, still has hydrolyzation activity even thought that is less than 5%. If the D204 starts the nucleophilic attack, the mutant should loss whole enzymatic activity. The remaining activity may imply it is an important assis- tant, or SMP-30 has alternative nucleophilic. Or non-specific hydrolyzation co-exists in the measurement. SMP-30 metal substitutional experiment [16, 18] showed that different functions were presented when different divalent metals were bound. According to results of Chakraborti et al., SMP-30 has gluconolactonase activity when it bound with Zn2+, Mg2+, Mn2+, Co2+ or Ca2+. Among those substituted enzymes, Zn2+ has the highest activity, as shown in

table 1.2. From the Kd values, manganese shows the best binding aﬃnity. However, because

6 Table 1.2: Kinetic characterization of SMP-30 with gluconolactone and its metal dependence [16]

−1 −1 −1 KM (mM) kcat(s ) kcat/KM (s mM ) Kd (µM) cellular concentrations Zn2+ 2.7 ± 0.5 341 ± 35 126 ± 27 7.0 ± 0.9 fM - nM Mn2+ 0.6 ± 0.1 79 ± 8 132 ± 26 0.6 ± 0.1 0.1 − 10 µM Mg2+ 1.3 ± 0.3 31 ± 4 24 ± 7 82 ± 21 500 µM Ca2+ 3.7 ± 0.7 48 ± 6 13 ± 3 566 ± 249 0.1 − 1 µM

in the human body, the concentrations of Ca2+ and Mg2+ are much higher than other divalent metal cations. Therefore, most of the found wild type is calcium-dependent or/and magnesium-dependent SMP-30. Overall, even though the metal substitution experiment didn’t reveal the entire mechanism, the results point out that SMP-30 active site is capable to chelate diﬀerent divalent cations, and this replacement shows that the metal has high potential to aﬀects the kcat values and KM values.

1.2.2 Diisopropyl ﬂuorophosphatase (DFPase)

DFPase [14, 17, 19, 20], another calcium-dependent protein in the N6P superfamily, also is an organophosphate hydrolase and lactonase. DFPase cannot hydrolyze paraoxon and parathion, but can hydrolyze DFP after calcium was bound to the active site. As shown in fig 1.3, PON1 and DFPase share similar topology and enzymatic functions. Especially, compared with SMP-30, DFPase and PON1 both have two calcium cation in their structures, one for structural stabilization and one functions as catalytic center. Both of them have five amino acids (E21, N120, N175, D229 and G230 in DFPase; E53, N168, N224, D269 and N270 in PON1 ) around the catalytic calcium. Considering two additionally binding water molecules, both of enzymes have seven ligands bind to the catalytic calcium with the distorted geometries. However, the amino acids for proton receptor and the structural holder locate in different positions. Therefore, it still may have some differences in mechanisms of PON1 and DFPase. Like other members in the N6P superfamily, two possible mechanisms of DFPase were suggested: amino acid (Asp229) as the nucleophile, or the activated water attacks the substrates (shown in fig 1.5, the water molecule was activated by Asp229, then attacks as a nucleophile). In order to figure out which mechanism is more possible, Blum [19, 20] addressed this question by using metal replacement, nuclear density mapping and two kinds of simulations. According to the simulation by Blum et al [20], the nucleophile in DFPase hydrolysis process may be Asp229, not water. In the active mode of their

Figure 1.6: Nuclear density map of DFPase active site [20]. Yellow lines indicate the ligand that direct chelate with calcium. Red lines rep- resent the hydrogen binding between two atoms. The orientation of water molecule is shown and the carboxyl group of Asp229 is deprotonated. Figure 1.5: Schematic representation of possible mechanisms for DFPase. (A) The scheme of direct hydrolysis by a nucleophilic residue D229 (B) The general base catalysis, which the water molecule is activated by Asp229 and attacks substrate. [19]. model, the water which binds to the calcium ion, is water molecule instead of hydroxide (fig 1.5(a)). In addition, Blum et al did the DFPase replacement in 2006 [19]. They pointed out that the Mg2+ and Ba2+ replacements of DFPase showed ∼5-20% higher activity relative to the wild-type (Ca2+). Connecting the simulation results with the metal replacement experiments, Blum et al [20] proposed one possible explanation for supporting the first mechanism(fig 1.5(A)): different metals in the active would cause varied energy boundaries. Similar concept was mentioned in the computational thermodynamic results from Katz et al. in 1998 [21]). The deprotonation energy of water will decrease ∼ 60 % if water-Mg2+ complex is form and metal activates the water. However, when Mg2+ bound with carboxylate groups (in DFPase are E21 and D229, as shown in fig 1.6), the free energy of deprotonation of water would be increased ∼ 80 kcal/mol, since the repulsion tend to attenuate the number of negative-charged ligands on one metal. Therefore, if the activated water attacking mechanism is correct, the change of catalytic activity should fit the trend of the different free energy of deprotonation.

1.3 Promiscuity of Organophosphatases

One of PON1’s deﬁnitive characteristics is promiscuity. The deﬁnition of promiscuity [22] is that one enzyme functions on more than one kind of substrates. For example, PON1 hydrolyzes organophosphates and lactones. Even though the natural substrates of PON1 is lactones, it still has decent activity of organophosphates. Because promiscuity is not a common feature of enzyme, several hypotheses were suggested for explaining the phenomenon.

8 Figure 1.7: (a) Simpliﬁed free-energy proﬁle comparing an idealized enzymatic one-step process with the corresponding background reaction, illustrating ground state (KS) and transition state (KTS) binding. (b) Cartoon representation of pocket of promiscuous enzyme. [22].

Based on thermodynamics, the energy diagram of the simulation are shown in fig 1.7. As can be seen, fig 1.7(a) shows the basic concept of enzyme. The hydrophobic pocket in the enzyme can create another distinct chemical environment, usually reducing the free energy of the specific chemical reaction. Therefore, it increases the possibility of chemical reaction happening and enhances the efficiency. fig 1.7(b), on the other hand, illustrates the conceptual binding pockets of promiscuous enzymes. Most promiscuous enzymes have less amino acids side chains in the binding pockets, so their pockets are capable of containing different kinds of substrates. In other words, only the charged or polar side chains will appear in the active site, which also provide the needed groups for enzyme mechanisms, such as electrostatic stabilization, proton acceptor/donor, or nucleophilic functional groups. To date, for promiscuous metalloenzymes, identifying which kind of promiscuities is the wildly-discussed question. There are two kinds of promiscuities: substrate promiscuity, which means enzyme has the same chemical reaction for different substrates; and catalytic promiscuity, which refers to that enzyme has different transition states are involved for different chemical reaction [22]. To address this issue, several methods are applied, one of them is metal substitution.

1.4 Metal Substitution

Metal substitution is one of the common approaches for understanding metalloproteins. Most metalloproteins have selectivity for metal cations choices. Several reasons are critical for proteins to select those cation, such as the concentration in the cells, ionic radii, surface potential, oxidation number and the geometry of empty/half-filled orbitals. Also, because different ligands will have divergent binding affinities with the metal center, metal selectivity also depends on the ligands that connect to the metal. Different combinations between

9 metals and ligands affect the electron distribution, which influences the binding of substrates and other molecules. The ligand orientation and the metal binding site can be gathered either from crystal structures or simulation results. Therefore, some details of mechanism can be provided. In addition, it is possible for metal substitution to provide some thermodynamics details. As mentioned in section 1.2.2, Mg2+ substituted was found less hydrolysis rate, and the possible hypothesis is the metal variant would cause the deprotonation energy differing. Another possible hypothesis is due to the divergent coordination numbers between Ca2+(8- fold coordination) and Mg2+6-fold coordination). It turns out that Mg2+ substituted does not have enough coordination for water to bind. Plus, several analytical tools such as EPR or fluorescence, ask for particular chemical or physical properties, and metal substitution can generate these properties such as using paramagnetic metal substitutes for diamagnetic metal.

1.4.1 Case Study: Phosphortriesterase

Phosphotriesters(PTE) [23–26] is one of the remarkable case for applying metal substitution with promiscuous organophosphate hydrolase from 90s [25]. PTE hydrolyzes phosphotriesters, thiophosphates and phosphorothiolates are analogs of phosphotriesters. PTE has the TIM barrel(α/β) structure with two zinc metals bound in the active site. Different then N6P superfamily member, the PTE hydrolyzation involves two zinc cations, shown in fig 1.8. The structure of PTE cause it’s promiscuity. It has three pockets with different sizes and hydrophobicities, those various pockets are capable for accommodating different substrates (showed in fig 1.8(b), with different colors). In the center of active site, these two zinc cations ligate to 6 amino acids. The coordination of α-metal is trigonal bipyramidal with two histidines (H55 and H57) and one aspartic acid (D301); β-metal has more solvent exposed, plus two histidines (H201 and H230). In addition, there is one amino acid with specific modification and chelates with both zinc cations, K169. K169 has carbamated and two C−O bonds work as bridge. To date, the potential mechanism explanation is shown in fig 1.9. After substrates binds to the β-zinc, the bridged water will attack the phosphorous center and a bridged phosphate is generated. Later on, two water molecules replaced the O−Zn bonds and release substrates. For establishing this hypothesis, scientists have done several metal substitutions and measured these substituted-PTE with MCD [27,28], EPR [29], NMR [30] and X-ray [31].

10 Figure 1.8: Key interactions of the Michaelis complex of paraoxon substrate and phosphotriesterase. The structure is taken from the last conﬁguration of the molecular dynamics simulations of the Michaelis complex state during the umbrella sampling calculations. (A) The QM subsystem displays the binding orientation between paraoxon and PTE active site. (B) Schematic illustration for PTE active site. [23].

Magnetic Circular Dichroism [27, 28, 32–34]

Magnetic circular dichroism(MCD) is widely used to monitor the ligand-field excited states. [32–34]. fig 1.10(a) shows the principle of MCD. In short, when there is an additional magnetic field, the degeneracy energy levels would have coupling with that field and the energy level will be shifted. And different orbitals have different tendencies of different polarized incident lights because of the selection rules. Therefore, the molecules have differential absorption of left and right circularly polarized electromagnetic wave. Overall, there are three different modes of MCD as shown in fig 1.10(b). Equation 1.1 is the overall MCD intensity [34]. There are three different kind of MCD absorption, A-term,

11 Figure 1.9: The suggested mechanism for phosphortriesterase [24].

Figure 1.10: (a) A schematic of the MCD experiment. (b) Energy level diagrams of A-, B- and C- term.

B-term and C-term, correspond to three modes in ﬁg 1.10.

∆ ∂f (E) C = γB −A + B + 0 f (E) (1.1) E 1 ∂E 0 T In Equation 1.1, ∆ is the molar absorptivity diﬀerence between left and right circularly polarised light, E is energy of incident radiation, γ is the constant and f (E) is the bandshape function. For metalloproteins, the asymmetric ligands cause non-degenerate ground states. When there is a paramagnetic metal in the active center such as iron or copper, metal itself has spin degeneracy. So after metal and ligands coupling, the degeneracy of the molecular

12 Figure 1.11: Summary of ligand field calculations in MCD spectra [27] orbitals will slightly regain and C-term MCD signal can be detected [34]. Ely et al. [27, 28] substituted Zn2+ with Co2+ in PTE (OpdA in the article), and the results are shown in fig 1.11. As can be seen, after replacement, the coordinations of α- and β- metals can be found. Combined with the structural information, α-metal is identified in a trigonal bipyramidal geometry. In addition, fig 1.11 shows that the MCD spectra changed during PTE-EPO reaction (OpdA-EPO). The geometry of α-metal remained the same, but the coordination of β-metal changed from six-coordination to five coordination. This result may support the mechanism in fig 1.9 that during the hydrolysis, the bridged hydorxide between α- and β metals will break the binding with β-metal and act as a nucleophile for attacking substrates.

X-ray Crystallography [35]

X-ray crystallography, without doubt, is one of the most powerful tools for determining metalloprotein structures. It can provide accurate spacial arrangement: the relative coordinations of every amino acid (at least in solid state), and the precise bond-lengths. Therefore, for metalloprotein identifications, x-ray crystallography can reveal the metal binding site and identity of ligand as well as its orientations and distances. Even though there are some disadvantages of X-ray crystallography for metalloproteins such as difficulty for assigning 13 electron density [36] and distinguishing the co-crystallization, X-ray crystallography still provides numbers of convincing information. PTE has [31] various crystals with different metal substitutions, such as Zn2+/Zn2+- , Zn2+/Cd2+-, Cd2+/Cd2+-, and Mn2+/Mn2+-PTE. Based on the results of Benning et al. in 2001 [31], two surprising phenomena were found by metal substituted PTE: (1) In Zn2+/Cd2+-PTE, they found an unpredicted carboxylated K169 since carboxylation did not be found in the apo-enzyme structure; (2) In the superimposed conformation of PTE within or without metal binding, the backbone conformations (especially Asp 301) are deviated (root-mean-square 3.4A).˚ For other metal substitution models, a β-metal was found that has 6-coordination in Zn2+/Cd2+-, Cd2+/Cd2+-, and Mn2+/Mn2+-PTE. However, Zn2+/Zn2+- PTE (wild type) was found to have a five-coordination β-metal. These two kinds of results fit the outcome in MCD (section 1.4.1).

Electron paramagnetic resonance (EPR) [29, 37]

Electron paramagnetic resonance (EPR) is a technique for analyzing molecules which contain unpaired electrons [38]. The basic concept is similar with MCD. The external magnetic field splits the energy levels according to the spin directions of elections. Different ligands would create different ligand field and which generates different absorption spectra. For biological compounds, the main applications are (1) measuring the transition metals and radicals in biological molecules; (2) studying environment and dynamics of the radical labelled compounds; and (3) using a radical trap to follow electron transfer. For PTE, Samples et al. [29] used Mn2+-PTE to comprehend the dynamic process of PTE hydrolysis. Samples et al. [29, 37] analyzed the hyperfine splitting of Mn2+-PTE, in which the spectrum would be affected if the binding ligand. They measurement the reaction at low temperature and monitor the slow signal change. As the results, they elucidated the protonation and deprotonation of the bridge hydroxyl by observing the g value changed, also adjust the pH value and confirming that the bridging hydroxide is the attacking nucleophile.

Nuclear magnetic resonance (NMR) [30, 39]

NMR is known to measure the radiation absorption by the nuclear spins and magnets of molecules when an additional magnetic ﬁeld applied. It is widely used for chemical compound determinations. However, if metalloprotein contains diamagnetic metal ions, it would be as normal as apo-enzyme; if it contains paramagnetic metal ions, it would aﬀect the range that NMR can monitor [39]. When there is a paramagnetic metal ion in the protein and the spin quantum number I greater than 1/2 will make other signals have poor resolution because of the quadrupolar moments. The signal of other atoms will be attenuated and cannot be identify easily. However, the right metal chosen can reduce

14 this effect. Among all of metals, 113Cd is considered to be the one has good signal after substitutiton. Omburo et al. [30] applied 113Cd NMR since 113Cd ( a nuclear spin of 1/2) has some benefits such as the chemical shift is sensitive to coordination environment and the shift is significant. Because of those benefits, it can provide the information of metal-protein, metal-substrate and metal metal interactions. The 113Cd NMR results showed that the chemical shifts of α- and β-metal are diverged (∼100 ppm, 161 ppm and 212 ppm individually), which implies that there is a notable difference between these two metal binding sites. In addition, the binding rate of α-metal is faster than β-metal can be found during apo-PTE is titrated with Cd solution. Plus, when adding Cd2+ solution into Zn2+-PTE, the result shows there is a competitive replacement: α-metal binding site tend to maintain Zn2+, and β-metal binding site can be substituted.

1.4.2 Other Methods for Metalloprotein Analysis

Raman Spectroscopy [40]

Using Raman for metalloenzyme research has some advantages: (1) Raman spectroscopy is capable of measuring in aqueous solution; (2) Resonance Raman spectroscopy can reduce the interfere from the scattering bands since it stimulates the electron with speciﬁc light wavelength; (3) The intensity of Resonance Raman is sensitive to the environment and molecular conﬁgurations, so mechanism monitor may be feasible.

Lanthanide Fluorescence [41, 42]

Lanthanides have different electron configurations and based on that, it is possible to generate different energy levels. Since 4f orbitals, where lanthanides are filling in with electrons, are not outer orbitals, the valence shell electron configuration, 6s2, will remain the same. Moreover, since the 4d orbitals are inner orbitals, the trends in ionization energies and ionic radii of lanthanides are smooth and steady. In contrast, the high density of protons in the nucleus causes that the radii of lanthanides become smaller. Scientists use lanthanides as substitutions for calcium ion in biological research because radii of lanthanides are close to the radius of calcium ion. For biochemical research, the T1 level of tryptophan are usually slightly higher than the LUMO of terbium [42], which means after intercrossing between S1 state and T1 state, an electron can transfer to LUMO of terbium and fluorescence from LUMO to HOMO of terbium, as shown in fig 1.12.

15 Figure 1.12: Energy diagram of the lanthanide complex, consisting of a sensitizer and a lanthanide(III) ion. A good sensitizer such as tryptophan (Trp) [42]

PAGE-LA-ICPMS [43]

The combination of polyacrylamide gel electrophoresis (PAGE) and laser-ablaton(LA)- ICP-MS can probe the element content of protein. Laser can ablate specific proteins on the gel. The advantages for using PAGE-LA-ICP-MS includes its speed and efficiency, and no purification process is needed.

Figure 1.13: Images of Zn, Cu, Fe, Pb, Mn and Ag in 2D-blue native gel electrophoresis gel of rat kidney water extract [43]

PAGE-LA-ICP-MS a uses laser beam to specifically measure the element distributions on the gel. As shown in fig 1.13, Becker et al. [43] analyzed the Cu2+-exchanged bovine serum albumin proteins with gel and did the measurement of different kinds of element

16 traces. The distribution of Zn2+, Cu2+ and other metals are clearly shown. After matching with the protein distribution (by MALDI or other methods), the metal component of protein can be determined.

1.5 Previous Metal Substitution in PON1

As mentioned above, metal substitution can provide supporting information for understanding metalloproteins such as conformations and mechanisms. Therefore, Several metal-substitution relevant experiments have been done on PON1 in the past [3,11,44–47]. Kuo et al. [9] examined the eﬀects of metal selection in the PON1. They dialyzed PON1 in the low calcium concentration buﬀer (∼ 10−8 M) for 18-24 hr at R.T.. As the results, two key conclusions about metal study are made. First, Zn2+, Cd2+, Co2+, Mn2+, Sr2+, Ba2+ and Mg2+ can sustain PON1 in the active form with nearly zero activities (Data not shown in the paper). To the contrary, Au2+, Hg2+ and Cu2+ can not maintain active form. And second, adding Ca2+ solution to the apo-PON1 can fully regain the activity. Even though their results have demonstrated some properties of PON1 metal center, the methods they used may remove both structural Ca2+ and catalytic Ca2+. In this case, the result can be determined by both the selections of structural or catalytic binding sites.

Figure 1.14: Terbium-PON1 fluorescence emission spectrum. Purified human PON1 (5 M) in 20 mM MES/NaOH buffer, pH 6.5, containing 0.1 mM CaCl2, 0.1 mM TbCl3, and 20% glycerol was excited at 282 nm. A 355 nm short wavelength cutoff filter was used in the emission path to reduce the intense protein fluorescence emission as a consequence of the 282 nm excitation. The four maxima at 489, 545, 585, and 620 nm are characteristic of Tb3+ emission, sensitized after an energy transfer from a nearby aromatic amino acid.). [3] 17 Figure 1.15: The inhibition patterns of wild-type-like rePON1 and of the H115W mutant 2+ 2+ 2+ by the divalent metal ions. (A) Mg (MgCl2), (B) Sr (SrCl2), (C) Cu (CuCl2) and 2+ (D) Ba (BaCl2). Shown is the residual paraoxonase activity ([S]0 = 2 mM), at various concentrations of competing metals, in the presence of 1mM CaCl2 (initial rate with no competing metal = 100%). [11]

Lockridge and her group replaced calcium with terbium and studied the mechanism of PON1 hydrolyzation with fluorescence-energy-transfer spectroscopy in 1999 [3]. As shown in fig 1.14, after adding high concentration terbium solution, fluorescence with 545 nm when PON1 is excited with 285 nm, which fit the theoretical prediction that there is a Try/Trp around the terbium binding site. Besides, PON1 lost it whole activity may also indicate that calcium is not at the active site. Lockridge et al. also replaced calcium with its isotope, 45Ca2+, then 45Ca2+-substituted PON1 was reacted with group-selective reagents. The result suggested that the Trp, Asp, or Glu amino acids covalently bind calcium in the active site . Ben-David et al. [11] also examined the competitions between calcium and other metals(Ba2+, Mg2+, Cu2+ and Sr2+) in PON1, the result is shown in fig 1.15. Except Sr2+, inhibition patterns are shown when other metal solutions had added. However, the inhibition patterns which is generated by additional metal solutions may due to two possible reasons. First of all, the catalytic calcium in PON1 may be replaced by the displacing metal, but the displacing metal does not have good hydrolysis potential as

18 calcium. Second, adding other ionic solutions may cause the change of ionic environment and aﬀect the function of PON1.

19 Chapter 2 Methods

2.1 Expression of Paraoxonase I

Expression and purification of PON1-G3C9 were follow the protocol in the previous research [11,48]. PON1 G3C9 with C-terminal 6-His tag was expressed in the BL21 cells with ◦ 2YT broth at 37 C until the OD600 value is between 0.6 and 0.8. Then, 0.1mM IPTG was added to induce culture for 4 hours at 30◦C, and the cells were centrifuged down and the pellet was gathered. Pellet was suspended in the lysis buffer with the ratio 50-60 ml lysisi buffer for 1 L growth for 30 mins, where lysis buffer had Tris pH 8 50 mM, CaCl2 1mM, NaCl 50 mM, BME(β- mercaptoethanol) 0.1 mM. The nuntating process was at 4◦C. After checking all cells were suspended, and PMSF(Phenylmethanesulfonyl fluoride) 1 µM was added, broke cells with sonication, and 0.1% tergitol was added right after sonication. The lysis solution was shaken at 4◦C for 2.5 hours, and the lysis solution was centrifuged at 10,000 RPM for 20 mins. Then the supernatant was transferred to an- Figure 2.1: The gel of PON1 G3C9. other clear tubes for purification. L is ladder, P is pellet, FT is flow through, W1, W2 and W3 are wash Ni-NTA slurry (4 ml resin, 50% slurry in EtOH; buffer gathered after colume, E1 and 8mL/1L growth) was taken and centrifuged 2 mins at E2 are elution buffer, which contain 1,000 RPM. Then the supernatant was removed and PON1 as label in red rectangular activity buffer (Tris pH 8 50 mM, CaCl2 1mM, NaCl frame. 50 mM, Tergitol 0.1%) was added to wash Ni-NTA resin. The washing process were repeated three times. Washed Ni-NTA was added into the supernatant-containing tube. The tube was in-

20 cubated in shaker for 3 hours at 4◦C, then the solution was put into the washed column and let unbound protein flow through. The column was washed by wash buffers (W1 and W2: 50 ml activity buffer with 10 mM imidazole; W3: 20 ml activity buffer with 25 mM imidazole) and elution buffers(E1 and E2: 30 ml activity buffer with 125 mM imidazole). Elution buffers were gathered and PON1 enzyme would be harvested. Fig 2.1 is the gel of PON1. In order to store PON1, the gathered PON1 enzyme was dialyzed with 1L 10% glycerol solution (50 mL 1M Tris-HCl pH8, 1 mL 1M CaCl2 , 5 mL 5M NaCl, 10 mL 10% tergital and 100 mL pure glycerol) and 1L 50% glycerol solution (50 mL 1M Tris-HCl pH8, 1 mL 1M CaCl2 , 5 mL 5M NaCl, 10 mL 10% tergital and 500 mL pure glycerol) at 4◦C. The period of each dialysis process was 2 hours. The concentrated PON1 would be stored at -20◦C.

2.2 Bradford Assay

All the protein concentration determinations in this work were brieﬂy scanned by routine Bradford assay. Bradford assay [49] is a high sensitive way to identify the concentration of protein within the range 1-50 µg [50]. This method uses Coomassie Brilliant Blue G- 250 as a dye (absorbance maximum at 466 nm and 650 nm [51]) to interact with arginine, histidine, phenylalanine, tryptophan and tyrosine residues in acidic siutation [52]. After the reaction is done, the dye-protein complex would have maximum absorbance at 595 nm [51]. After the concentration of complex is gathered, using Beer’s law, the linear range and the standard, the unknown concentration of protein can be found out. Firstly, Coomassie Brilliant Blue G-250 was diluted into 20% by deionized water, ﬁl- tered and stored at 4◦C. Bovine serum albumin (BSA) solutions were prepared in varied concentrations and used as a standard [51]. Microarray plate was taken. 200 µL Coomassie Brilliant Blue R-250 and add 10 µL protein solution was mixed in each wall of microarray plate. Waiting for totally reacting in about 5-10 mins and the color shift could be observed from orange to blue. Then the UV absorbance was measured at 595 nm. In order

to make sure the concentration was in the linear range of Bradford assay, the OD600 value should be between of 0.6 and 0.7. After standard (BSA) had measured, the same processes was repeated with PON1 solutions and the absorbance was gotten, the concentration was interpolated.

2.3 Apo-Paraoxonase I - Dialysis Processes

Apo-PON1 G3C9(dilute to 1mg/mL) was gathered by dialyzing PON1 solution in the EDTA solution for 1 hr, which had 50 mM Tris pH8 buﬀer, 50 mM NaCl and 50 mM EDTA. Then, in order to reduce the EDTA concentration in the dialysis bag, dialysis bag

21 was switched to 1 mM EDTA, 50 mM Tris pH8 and 50 mM NaCl buﬀer for 30 minutes. The fresh 1 mM EDTA buﬀer was replaced three times to ensure the concentration of EDTA in dialysis bag was close to 1 mM. The PON1 enzyme after EDTA treatment was stored in 4◦C.

2.4 Kinetic Measurement

Table 2.1: The list of substrates and extinction coeﬃcient

Extinction Substrate Reference Coeﬃcient (ε, Structure M −1cm−1)

Paraoxon(Px) [53] 17,100

Chlorpyrifos oxon(CPO) [54] 5,560

Dihydrocoumarin(DHC) [55] 876

Phenyl Acetate (PA) [53] 1310

5-thiobutyl [56] 7000* butyrolactone(TBBL)

*The absorbance is from 2-nitro-5-thiobenzoic acid(TNB), released from DTNB, the probe of TBBL

All processes of kinetic measurement were mentioned in the previous research [48]. Firstly, 186 µL buffer (50 mM Tris ·HCl buffer, 50 mM NaCl) was mixed with 4 µL substrates. Secondly, the 5 µL enzyme was mixed with 5 µL 10 mM cation solution; for negaitve control, buffer solution is added instead. Thirdly, 10 µL mixed enzyme solution was added into the wall, totally mixed with the substrates and sent into the spectroscopy immediately. The spectroscopy for real-time enzyme kinetic measurement we used was SpectraMax M5 Microplate Reader (Molecular Devices, LLC.; USA).

22 Three response variables in this experiment are designed: pH value of the buffers, replaced cations and substrates. Firstly, based on previous review [57], PON1 has found to have good activity in the pH value range between 7.5 and 8.5. PON1 activity will be gone when the pH value lower than 7, and hydrolyzation when the pH value is greater than 9. Therefore, in this experiment, pH values were between 6 and 9, with 0.5 interval in the analysis. Secondly, the substituted cation were magnesium(II), calcium(II), manganese(II), iron(III)/iron(II), cobalt(II), nickel(II), copper(II), zinc (II), strontium(II), terbium(III) and europium(III). Because those cations are common metals in metalloproteins, they are chosen for understanding the selectivity of PON1 metal binding. Also, according to table 4.2, iron(III) may partially reduced to iron(II) in acidic environment(standard reduction potential is 0.77V). Thirdly, table 2.1 lists all the substrates that were tested in this work. As mentioned in the section 1.3, the promiscuity of PON1 may imply there is two mechanisms of PON1 for hydrolysis of organophosphates and lactones. Therefore, substrates in table 2.1 were used for investigating the effect of different kinds of substrates.

2.4.1 5-thiobutyl butyrolactone(TBBL) Measurement

Dr. Tawfik and Khersonsky [56] demonstrates how to measure hydrolysis rate of PON1 by TBBL in previous research. 5,5-dithio-bis-2-nitrobenzoic acid(DNTB, also called Ellman’s reagent). Hydrolysis of TBBL does not make the absorbance difference; Hence indicator is needed for detecting. Fig 2.2 shows the mechanism of DNTB. As can be seen, after TBBL is hydrolyzed, hydrolyzed product, 4-(butylthio)-4-hydroxybutanoate, would spontaneously break down and generate 4-oxobutanoate and butane-1-thiolate. butane- 1-thiolate would attack DTNB, break disulfide bond and release TNB2–, which has an absorbance at 412 nm and works as the indicator.

Figure 2.2: Mechanism of DNTB as23 indicator of TBBL hydrolyzation [56] 2.4.2 Paraoxon and Dihydrocoumarin Measurement

Paraoxon(Px) and Dihydrocoumarin(DHC) are known that would decompose and aﬀect the concentration. Therefore, in order to ensure the concentration of substrates, chemical hydrolysis measurements are done before the kinetic measurement. For checking the hydrolysis situation of the Px, 48 µL Px was mixed with 952 µL methanol. According to the labeled concentration on the bottle, the ﬁnal concentration should be around 130 mM. Then two groups of tests were prepared. For chemical hydrolysis group (experiment group), 2 µL 130 mM Px was mixed with 2 µL 5M NaOH and 176 µL

H2O; For non-hydrolysis group (control group), 2 µL 130 mM Px was mixed with 178 µL

H2O. After overnight, 20 µL 10 times concentrated assay buﬀer was added into the solution in both group, then make 1:100 dilution of both solution. 200 µL solution of each group was taken and measured the UV absorbance at 412 nm. Using the absorbance of NaOH group as the total hydrolyzed concentration, we could deduce the hydrolyzation level of control group, which was equal to the concentration of the stock. Likewise, for DHC hydrolysis situation checking, 6.34µL DHC (7.89 M) was mixed with 933.66 µL methanol. Theoretically, the ﬁnal concentration should be around 55 mM. Then two groups of tests were prepared. For chemical hydrolysis group (experiment group), 55 mM DHC was diluted 104 times into 0.53 mM. 100 µL 0.53 mM DHC was mixed with 100 µL 0.5 M NaOH. Solutions were incubated in 37◦C for 10 minutes. UV absorbance of solutions were measured 270 nm. For standard 0.53 mM DHC solution, OD270 should be 0.284.

2.5 Calculation

Figure 2.3: Calculation process

In the kinetic measurements, all the calculation process were shown in Fig 2.3. According 24 to Beer-Lambert law (equation 2.1) and table 2.1, the absorbance is proportion to the ∂A concentration of hydrolyzed products. As can be seen, after the slope of hydrolysis rate( ∂t in equation 2.2) was gotten, and divided by extinction coeﬃcient () and the path length ∂c (b), the rate of hydrolysis could be obtained( ∂t in equation 2.3).

A = bc (2.1)

∂A ∂c = b (2.2) ∂t ∂t

1 ∂A ∂c = = v (2.3) b ∂t ∂t 0 According to Michaelis–Menten kinetics (equation 2.4)

Vmax [S] v0 = (2.4) KM + [S]

where Vmax = kcat [E]total and [E]total = [E]0 = [E]t + [ES]. Therefore, when the [S] KM ,

[E]0 ≈ [E]t (2.5)

[S] [S] ≈ (2.6) KM + [S] KM Therefore, in the diluted substrates condition, Michaelis–Menten kinetics can be simpli- ﬁed into equation 2.8.

kcat v0 = [E][S] (2.7) KM

v k 0 = cat (2.8) [E][S] KM Consequently, after time unit conversion, the slopes were divided by the concentrations of substrate and enzyme in the wall, and the speciﬁc activity (kcat/KM ) would be gotten.

25 Chapter 3 Results

3.1 EDTA treatment of Paraoxonase I

Figure 3.1: The eﬃciency of EDTA dialysis. The speciﬁc activity is the activity of hydrolyzing paraoxon

As described in section 2.3, purified PON1 was firstly dialyzed with 50 mM EDTA solution. Fig 3.1 shows the change of PON1 activity to Px with different dialysis period. As can be seen, after 5 mins dialysis, whole PON1 activity was gone. This result indicates that the catalytic calcium of PON1 had been completely removed. In order to reduce excess EDTA in the solution, multiple rounds of 1mM EDTA dialysis solutions were used to ensure that concentration of EDTA of outer dialysis buffer would be

26 Figure 3.2: The eﬃciency of EDTA dialysis. The speciﬁc activity is the activity of hydrolyzing paraoxon

nearly constant. In other words, after 1 hr 50 mM EDTA dialysis, PON1 was switched into the fresh 1 mM EDTA and dialyzed 30 mins three times (”3 round” in fig 3.2). Similar, four round means PON1 was dialyzed in fresh 1 mM EDTA four times. Base on fig 3.2, after three rounds 1 mM EDTA dialysis, 10 mM calcium solution would let apo-PON1 regain the PON1 activity. Consider the kinetic effect, different concentrations of Ca2+ solutions were added and results are shown in fig 3.2. Mixed PON1 and calcium solution(stock concentration, final concentration is 2.5 mM) was placed for 24 hours, and activity was remeasured. As can be seen, the PON1 activities only had small fluctuation, which means rate of calcium rebound is relatively quick. This result may also imply that the calcium binding of PON1 is relevant to thermodynamics rather than kinetics. Later on, the substituted metal solutions were mixed with apo-PON1. After 2, 4 and 6 days incubations in 37◦C, metal-substituted PON1’s specific activities of hydrolyzing Px and DHC were measured. Fig 3.3 and fig 3.4 show that metal-substituted PON1s are stable in days. One thing needs to be noticed is the similarity between Fe3+-PON1 and Fe2+-PON1. 3+ 3+ – 2+ ◦ 3+ Since the reduction potential of Fe is 0.77V (Fe + e Fe E = +0.771V ), Fe will be partially reduced to Fe2+ when adding in the aqueous solution. After 2 days incubation, both Fe3+ solution and Fe2+ reach to the same chemical equilibrium. Therefore, in the fig 3.3 and fig 3.4, the specific activities of Fe3+-PON1 and Fe2+-PON1 are very close to

27 Figure 3.3: The speciﬁc activities of hydrolyzing Px with metal-substituted PON1. The measurement are done 2, 4 and 6 days after addition of substituted metal solutions

Figure 3.4: The speciﬁc activities of hydrolyzing DHC with metal-substituted PON1. The measurement are done 2, 4 and 6 days after addition of substituted metal solutions

each other. Because in the chemical equilibrium situation, Fe2+ is the majority in the solution, following discussion will mainly focus on Fe2+-PON1.

28 3.2 Substitution Other Divalent/Trivalent Metal Ion for Cal- cium Ion

After verifying cation can be stably rebound into PON1, the activity measurement was done. Fig 3.5 shows the result of hydrolysis rates with different substrates, when PON1 bound substituted catalytic metal cations. The 100% in the fig 3.5 is set as the specific activity of the original PON1, which means the natural activity before EDTA treatment. All the information is the ratio between the metal rebound PON1 and original PON1.

Figure 3.5: Activity of metal substitution at pH 7.5

Four substrates in the fig 3.5 have mentioned in table 2.1. Paraoxon(Px) is the organophosphate with a tetrahedral structure; 5-thiobutyl butyrolactone(TBBL), is the natural substrates of PON1, has a restricted five-member ring; Phenyl acetate (PA) is the lactone which has tetrahedral conformation; Dihydrocoumarin(DHC) is the lactone with flat structure. These four compounds generally cover the comparisons on both categorical and structural dimensions. For the metal substitution part in fig 3.5, rebound Ca2+ is the positive control and none represents non-metal buffer was added, as the negative control. The transition metals are trace metal that usually found in the metalloproteins: manganese(II), iron(II)/iron(III), cobalt(II), nickel(II), copper(II) and zinc(II), for sorting out the trend. Magnesium(II) and strontium(II) are the comparison with calcium since they are all in the IIA group, they

29 share the same outer electronic configuration and only change the ionic radii. Terbium(III) and europium(III) are known to have the similar ionic radii and function as a good calcium replacement for bioinorganic chemistry for a long time [41], they may generate fluorescence when there are sensing amino acids around, such as tryptophan or tyrosine. Therefore, they may be applicable for further research. For original PON1, as the standardized scale of comparison in fig 3.5 and fig 3.6, is stored in the calcium free buffer. After 2 days, the catalytic calcium would dissociate and the activity would be degraded. Therefore, activity over 200% could be found.

3.2.1 pH value Factor

The PON1 activity is highly dependent on the pH environment, as mentioned in section 2.4. The pH environment aﬀects both the stability of PON1 and the concentration of nucleophile, the water molecules or hydroxide groups. Fig 3.6 shows the speciﬁc activities of metal-substituted PON1s at pH 9.0.

Figure 3.6: Activity of metal substitution at pH 9.0

3.2.2 Metal Factor

In ﬁg 3.5, most of metal substituted PON1 still have hydrolysis capability. This result may indicate PON1 has good tolerance of catalytic metal in the binding site.

30 In main group metals, the trend between fig 3.5 and fig 3.6 are similar. Ca2+ is the greatest, follows by Sr2+ and Mg2+ is the weakest one. In the transition metal series, Mn2+, Fe2+ and Cu2+ show relative good activities, Co2+ only shows around 20% activity, but Ni2+ and Zn2+ shown rarely activities. As a comparison, in fig 3.6, activity of Co2+, Zn2+ and Eu3+ are rising.

3.3 Negative Control - Hydrolysis Rate of Pure Metal Solu- tion

One concert of this kinetic examination is that the cation hydrolysis. If the aqueous n+ complex (M(H2O)6 ) could hydrolyze substrates, then it should be taken as background. Fig 3.7 and fig 3.8 show Px and DHC hydrolysis results of calcium free buffer with other pure substituted metal solutions. In fig 3.7 and fig 3.8, the hydrolysis rates of pure metal solutions are so low that would not affect the results.

Figure 3.7: Negative control - Hydrolysis rate of Px with additional cation solutions

Figure 3.8: Negative control - Hydrolysis rate of DHC with additional cation solutions 31 3.4 Negative Control - Bovine Serum Albumin(BSA) Mea- surement

Another concert of this kinetic examination is that the surface charge of protein may also cause the non-specific hydrolysis. Most of the proteins have charges distributed on the surface for increasing the solubility in aqueous solution. However, these charges may also trigger hydrolysis as long as it has proper conformation and hydroxide groups around. Fig 3.9 and fig 3.10 show the rate of hydrolyzing Px and DHC with BSA as a comparison for non-specific hydrolyzation, since BSA is not an organophosphatase. Fig 3.9 and fig 3.10 prove that the surface charge of BSA would not generate the same hydrolysis rate as metal- substituted PON1s.

Figure 3.9: Negative control - Hydrolysis rate of Px by BSA with additional cation solutions

Figure 3.10: Negative control - Hydrolysis rate of DHC by BSA with additional cation solutions 32 Chapter 4 Discussion

The properties of metal are ﬁrst discussed, such as pKa, reduction potential, binding constant and the lability of metals, in order to analyze the relationship between substituted metals and the hydrolysis rate.

1 2 3 E + S ES EP E + P (4.1) Fundamental enzymatic reactions can be separated into three steps (eq. 4.1). In the step 2, the hydrolysis tendency is affected by the affinities between metal and residues/water molecules (in sec 4.1, 4.2, 4.3, 4.4); Step 1 and step 3 are influenced by the affinity between metal and substrates(in sec 4.1); Whole stability of metal-substituted PON1 is affected by the binding affinity between metal and ligands (in sec 4.1). However, they all share the similar trends.

4.1 Metal-Ligands/Water Molecules Aﬃnity

n+ (n−1)+ + [M(H2O)6] + H2O [M(H2O)5(OH)] + H3O (4.2)

pKa (eq. 4.2, table 4.1) refers the ability of substituted metal for dissociating water and producing hydroxide group. In the general base catalytic hydrolysis (alternative Worek’s mechanism in ﬁg 1.2), the water molecule/hydroxide group directly attacks the center phosphorous and triggers the hydrolysis process. Theoretically, metals with lower pKas can easily generate hydroxide groups in the same environment. Therefore, since hydroxide group is a stronger nucleophile than water, metals which have lower pKas should preform better in general basic catalytic hydrolysis.

However, the results show an opposite tendency, in which the metal with higher a pKa value, substituted-PON1 shows better hydrolysis rate for substrates. The reason of this phenomena is not totally understood.

One possible explanation is that at lower pKa values metals have strong connections with oxygen atom in the water molecule, weakening the O−H bond and increasing the 33 Table 4.1: Experimentally determined pKa value and calculated deprotonation energies for the d-block metal complexes [58]

−1 Metal complexes pKa ∆ E kcal mol Ref. 2+ [Ca(OH2)6] 12.6 21.6 [59] 2+ [Sr(OH2)6] 12.4 [59] 2+ [Mg(OH2)6] 12.3 23.7 [59] 2+ [Mn(OH2)6] 10.6 42.3 [58] 2+ [Ni(OH2)6] 9.9 45.6 [58] 2+ [Zn(OH2)6] 9.5 41.3 [58] 2+ [Fe(OH2)6] 9.5 38.3 [58] 2+ [Co(OH2)6] 8.9 35.8 [60] 2+ [Cu(OH2)6] 6.8 5.0 [60]

n+ Table 4.2: Standard Reduction Potentials of M(OH)n and M Half reaction E◦/V – – Ca(OH)2 + 2 e Ca + 2 OH - 3.02 – – Sr(OH)2 + 2 e Sr + 2 OH - 2.88 – – Mg(OH)2 + 2 e Mg + 2 OH - 2.690 – – Mn(OH)2 + 2 e Mn + 2 OH - 1.56 – – Zn(OH)2 + 2 e Zn + 2 OH - 1.249 3– – 2– – PO4 + 2 H2O + 2 e HPO3 + 3 OH - 1.05 – – Fe(OH)2 + 2 e Fe + 2 OH - 0.88 – – Co(OH)2 + 2 e Co + 2 OH - 0.73 – – Ni(OH)2 + 2 e Ni + 2 OH - 0.72 – – Cu(OH)2 + 2 e Cu + 2 OH - 0.222 Half reaction E◦/V 2+ – Sr + 2 e Sr - 2.899 2+ – Ca + 2 e Ca - 2.868 2+ – Mg + 2 e Mg - 2.372 3+ – Tb + 3 e Tb - 2.28 3+ – Eu + 3 e Eu - 1.991 2+ – Mn + 2 e Mn - 1.185 2+ – Zn + 2 e Zn - 0.7618 2+ – Fe + 2 e Fe - 0.447 2+ – Co + 2 e Co - 0.28 2+ – Ni + 2 e Ni - 0.257 2+ – Cu + 2 e Cu + 0.3419 3+ – 2+ Fe + e Fe + 0.771

34 Table 4.3: Stability Constants (log K1) of Various Metal Chelates

Ligand(right) Aspartic acid Glutamic acid Pyrophosphate Succinic acid Metal(below) Ca 1.16 1.43 5.0 1.20 Mg 2.43 1.9 5.7 1.2 Sr 1.48 1.37 0.9 Mn 3.74 3.3 2.11 Fe(III) 22.2 3.28* Fe(II) 4.6 -2.65* Co 5.9 5.06 2.08 Ni 7.12 5.9 5.8 2.36 Cu 8.57 7.85 6.7 3.3 Zn 2.9 5.45 8.7 1.78 * is the thermodynamic stability constant from Gorman, J. E. [61]

possibility of proton dissociation. In other words, this metal is stable when it binds to hydroxide group. In the model of PON1 hydrolysis, after water has activated by either H115 (Tawfiks model, fig 1.2) or D269 (Alternative Woreks model, fig 1.2), the hydroxide group is released. In this case, the substituted metal that has lower pKa would tighter bind to the releasing hydroxide. Therefore, the hydroxide group would have less chance to attack phosphorus center. This trend of metal-hydroxide binding affinity also is supported by the reduction potential of metal in table 4.2, and the binding constants of metal and residues in table 4.3. The trend of metal-ligand binding affinity also can be explained with the similar concept. Aspartic acid and glutamic acid are the residues in the active site and bind with the center metal. It can be seen from table 4.3 that all substituted-PON1 with better hydrolysis capabilities have lower binding ability with ligands (aspartic acid and glutamic acid). This trend implies the threshold of hydrolysis is increasing with metal-ligand bond strength. Decreasing lability of ligands reduces hydrolysis. The concept corresponds with the effect of pKa.

Base on the pKa value and the standard reduction potential, the trend of metals having greater hydrolysis capability is following Cu2+ < Fe2+ < Ni2+ ≈ Co2+ < Zn2+ < Mn2+ < Mg2+ < Sr2+ < Ca2+ and the trend of experimentally observed data is Zn2+ ≈ Ni2+ ≈ Co2+ < Cu2+ ≈ Mg2+ < Fe2+ < Mn2+ < Sr2+ < Ca2+ As can be noticed, the trend of main group metals are similar, but the trend of transition metal does not. This difference indicates there are other factors (aside from metal-ligand binding affinity) affect hydrolysis rate. 35 4.2 Transition Metal - Irving-Williams series (pH7.5)

The Irving-Williams series [62] refers to the stabilities of high-spin complexes of divalent transition metals. Variables that inﬂuence the ranking includes ionic radii, crystal ﬁeld stabilization energy and geometry(octahedral).

Irving-Williams series: Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+

Base on the previous assumption, substituted metals with better stabilities would reduce the hydrolysis rate bonding breaking is more unfavored. Therefore, theoretical hydrolysis rate base on Irving-Williams series will be:

Zn2+> Cu2+ < Ni2+ < Co2+< Fe2+ < Mn2+

Combining with the trend with main group metals, theoretically, overall hydrolysis trend will be following and almost ﬁt the experimental observations (except Cu2+and Mg2+):

Zn2+> Cu2+ < Ni2+ < Co2+< Fe2+ < Mn2+< Mg2+ < Sr2+ < Ca2+

3– Actually, Irving and Williams [62] pointed out that PO4 this kind of conjugate base of strong acid may not following the trend since the metal-ligand interaction is weaker than metal-water interaction.

4.3 Dissociative and Associative Mechanisms

From the experimental observations, the metal-substituted PON1s with higher hydrolysis rates have a common property: these metals tend to have associative configuration change [60]. The concept of associative and dissociative hydrolysis mechanism can be illustrated by fig 4.1. It shows the tendency of having bond breaking first or bond making first. ∗ Thermodynamically, the metal with (1) greater ionic radii and (2) lower t2g and eg orbital occupancy would be on the associative side. The greater ionic radii have better extent, increase possibility of bond making and reduce the steric crowding. The lower t2g orbital occupancy would lower the repulsion force when solvent molecules approaching. The lower ∗ eg orbital occupancy will reduce the chance for d-electron attack to the solvent and break the metal-solvent bond. Ca2+, Sr2+, Mn2+ and Fe3+ prefer associative mechanism(data not complete shown). Fe2+ and Cu2+ are intermediate between two mechanism, depend on the substrates and ligands. Especially, Cu2+would be affected Jahn-Teller distortion. Jahn-Teller distortion shortens the metal-ligand bonds of four ligand in the equatorial plane and extends the

36 Figure 4.1: Interpretation of activation volumes for water exchange on hexaaqua metal ions in terms of contributions from bond making and bond breaking: Swaddles scaled More- OFerrall diagram assuming a continuum of interchange mechanisms [60] two axial ligand bondings. Therefore, Cu2+ has better activity since it may have great overlapping due to the distortion. Zn2+ and Mg2+, on the other hand, due to the small ionic radii and full orbitals, they tend to undergo dissociative mechanism more [60]. This common property supports the previous study that PON1 may have an associative hydrolysis. It also agree with the simulation model of Sanan et al. [10], in which catalytic calcium has an 8-fold coordination.

4.4 Transition Metal - Ligand Exchange Rate (pH9.0)

z+ ∗ kex ∗ z+ [M(solvent)n] + n(solvent ) −−→ [M(solvent)n−1(solvent )] + n(solvent) (4.3) For the pervious section, if PON1 hydrolysis process is associative, the rate of hydrolysis will be affected by the water exchange rate. Equation 4.3 defines solvent exchange rate. The small exchange rate indicates that the binding between water molecule and metal is stable and inert. Fig 4.2[60] shows the characteristic rate of H 2O -substitution in the inner coordination sphere of metals. The trend of substituted metals of water exchange rate (fig 4.2) is as follows: 37 Figure 4.2: Mean lifetimes, τ , of a particular water molecule in the first coordination H2O shell of a given metal ion and the corresponding water exchange rate k at 298 K. The H2O filled bars indicate directly determined values, and the empty bars indicate values deduced from ligand substitution studies. [60]

Ni2+ < Co2+ < Fe2+ ≈ Mn2+ < Zn2+< Cu2+

This trend agrees with the pH 9.0 result. This indicates that in a basic environment, the hydrolysis rate is mainly inﬂuenced by the ligand exchange rate. Ligand exchange rate can aﬀect the stability between metal/water molecule and metal/substrate.

4.5 Metal-Substrates Aﬃnity

In step 1 and step 3 of equation 4.1, the hydrolysis rate will be affected by the binding situation between metal/substrates and the metal/water. The affinity here are discussed from two sets of values: the binding constants and the lability of metal. The binding constants of metals and substrate analogs are presented in the last two columns of table 4.3. Pyrophosphate and succinic acid have similar structures with substrates organophosphates and lactones. Pyrophosphate shows the preference of metal to phosphate groups, even the structures are different between pyrophosphate and organophos-

38 phates; Succinic acid, on the other hand, shows the preferences of lactones due to the carboxyl groups, especially the ring structure of the succinic acid(succinic anhydride). The overall trend of metal-substrates stability, even though there are some missing values, is that metal with greater stability constants would have smaller hydrolysis capabilities. One potential explanation is that when the stability constant is greater, it reduces the turnover rate of the substrate. Even though the hydrolysis is done, the produce can not leave active site easily. As the result the overall performance of the metal-substituted PON1 is degraded.

39 Chapter 5 Conclusion

The metal substitution experiment proves that PON1 had capable to tolerate different cations in the active site and hydrolyze substrates. Different cations, due to different chemical and physical properties, would have different performances. Hence, the flexibility of metal binding in PON1 may indicate the binding site is spacious and relative nonspecific. Overall, PON1 presented good activities with catalytic metal such as Ca2+, Sr2+, Mn2+, Fe2+, Mg2+ and Cu2+.

Figure 5.1: Solvent exchange processes: volume profiles connected to the transition states. [60] 40 The performances of metal substituted PON1 showed the preference of catalytic cation for PON1 was the cation with high pKa and lower stability constants of metal-ligand bind- ings. As mentioned in section 4.3, the previous research [60] has studied the trend of mechanism change for first row transition metals. When d-electrons increases and the ionic radius decreases, the mechanism of water exchange shifts from associative(or intermediated associative) to dissociative (or intermediated dissociative). This tendency is proved by a change of sign for the activation volumes, the concept is shown in fig 5.1. The only exception is copper. With Jahn-Teller distortion, copper’s complexes often show high lability. Overall, the metal substituting-PON1 results suggested that PON1 has associative(or intermediated associative) hydrolysis mechanism. To date, there are three suggested mechanisms of PON1 (fig 1.2):

• Dr. Tawfik et al. [63] proposed a general base catalysis mechanism for lactonase activity for PON1. The activated water molecule is triggered by H115/H134 dyad. One of evidences for this mechanism is that mutating H115 would reduce the lactonase activity of PON1 by 100- to 600-fold [11]. However, the organophosphate hydrolase activity is enhanced. Moreover, Ben-David et al. [11] has addressed on this issue. Ben-David et al. find the position of catalytic calcium will have a 1.8 A˚ shift in H115 mutant. Therefore, they propose that lactonase activity and organophosphatase activity in PON1 have different mechanism with different catalytic calcium position. In this hypothesis, lactonase activity can be categorized as an intermediate process, which means ligands binds and break at the same time. In this model, after H115 activates water molecule, hydroxide group attacks center phosphorous directly, no metallic coordination number change get involved. Therefore, the volume profiles of transition state would not have significant change, and not significant coordination number change during the process.

• Dr. Worek et al. [64] also postulate that D269 acts as a nucleophile and catalytic calcium stabilizes the negative charge on the phosphoryl oxygen. However, the distance between oxygen atom of D269 and the phosphorus atom is relatively far. Actually, both simulations by Hu et al. [65] and Ben-David et al. [11] show the distance between D269 and phosphorous center is around ∼ 3.6 A.˚ Moreover, no covalent intermediates of the enzyme with either substrates or inhibitors is captured. Besides, this model can be considered as a dissociative process. Dissociative process means during the hydrolysis, the lower coordinations number complex can be observed. In this model, D269 changes from bi-dentate ligand to mono-dentate and generate a configuration change. That is to say, the coordination number of metal reduced in the first step. 41 • Alternative Worek mechanism, the other possible mechanism based on results of Worek et al. [64], a general base catalysis mechanism. However, they assume D269/H285 dyad activate water molecules, instead of H115/H134 dyad in Dr. Tawfik’s model. Their model is based on the neutron-scattering structure of DFPase, which shows the coordination of an intact water molecule to the calcium ion. In other words, water does not become hydroxide since the coordination to the calcium ion alone is insufficient. Indeed, the pKa and reduction potential both agree with that the calcium would not convert water molecule to hydroxide group. This model, which tends to increase coordination number first, is an associative process. The water molecule binds to metal and increases the coordination number of metal. Then it is activated by D269 and attacks substrate, which reduce the coordination number and finishes the process. The result of metal substituting experiment supported this model.

As aforementioned discussion, the alternative Worek’s mechanism may be the right mechanism.

42 Bibliography

[1] CIK Harsch. Mutagenic Studies of Substrate Specificity and Stability of Paraoxonase-1. PhD thesis, The Ohio State University, 2011. Cited on page1. [2] Amir Aharoni, Leonid Gaidukov, Shai Yagur, Lilly Toker, Israel Silman, and Dan S Tawfik. Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proceedings of the National Academy of Sciences of the United States of America, 2004. Cited on page1. [3] Denis Josse, Weihua Xie, FrédériqueRenault, Daniel Rochu, Lawrence M. Schopfer, Patrick Masson, and Oksana Lockridge. Identification of Residues Essential for Human Paraoxonase (PON1) Arylesterase/Organophosphatase Activities. Biochemistry, 1999. Cited on pages1, 17, and 18. [4] Denis Josse, Oksana Lockridge, Weihua Xie, Cynthia F Bartels, Lawrence M Schopfer, and Patrick Masson. The active site of human paraoxonase (PON1). Journal of Applied Toxicology, 2001. Cited on page1. [5] Michal Harel, Amir Aharoni, Leonid Gaidukov, Boris Brumshtein, Olga Khersonsky, Ran Meged, Hay Dvir, Raimond B Ravelli, Andrew McCarthy, Lilly Toker, Israel Silman, Joel L Sussman, and Dan S Tawfik. Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes. Nature Structural & Molecular Biology, 2004. Cited on page1. [6] Mohosin Sarkar, Christina Keventzidis Harsch, George T Matic, Kathryn Hoffman, Joseph R 3rd Norris, Tamara C Otto, David E Lenz, Douglas M Cerasoli, and Thomas J Magliery. Solubilization and humanization of paraoxonase-1. Journal of lipids, 2012. Cited on page1. [7] Holly G Davies, Rebecca J Richter, Matthew Keifer, Clarence A Broomfield, Jason Sowalla, and Clement E Furlong. The effect of the human serum paraoxonase poly- morphism is reversed with diazoxon, soman and sarin. Nature Genetics, 1996. Cited on page1. [8] Moshe Ben-David, Mikael Elias, Jean-Jacques Filippi, Elisabet Duñach, Israel Silman, Joel L. Sussman, and Dan S. Tawfik. Catalytic Versatility and Backups in Enzyme Active Sites: The Case of Serum Paraoxonase 1. Journal of Molecular Biology, 2012. Cited on pages1 and2. 43 [9] Chung-Liang Kuo and Bert N La Du. Calcium Binding by Human and Rabbit Serum Paraoxonases Structural Stability and Enzymatic Activity. Drug Metabolism and Dis- position, 1998. Cited on pages2 and 17.

[10] Toby T. Sanan, Sivaramakrishnan Muthukrishnan, Jeremy M. Beck, Peng Tao, Carri- gan J. Hayes, Tamara C. Otto, Douglas M. Cerasoli, David E. Lenz, and Christopher M. Hadad. Computational modeling of human paraoxonase 1: preparation of protein models, binding studies, and mechanistic insights. Journal of Physical Organic Chemistry, 2010. Cited on pages3 and 37.

[11] Moshe Ben-David, Grzegorz Wieczorek, Mikael Elias, Israel Silman, Joel L. Sussman, and Dan S. Tawﬁk. Catalytic Metal Ion Rearrangements Underline Promiscuity and Evolvability of a Metalloenzyme. Journal of Molecular Biology, 2013. Cited on pages 4, 17, 18, 20, and 41.

[12] Aljo ˇsaBavec, Damijan Knez, TomaˇzMakovec, Jure Stojan, Stanislav Gobec, and Marko Goliˇcnik.Exploring the aryl esterase catalysis of paraoxonase-1 through solvent kinetic isotope eﬀects and phosphonate-based isosteric analogues of the tetrahedral reaction intermediate. Biochimie, 2014. Cited on page4.

[13] A. Pla, L. Rodrigo, A.F. Hernndez, F. Gil, and O. Lopez. Eﬀect of metal ions and calcium on puriﬁed {PON1} and {PON3} from rat liver. Chemico-Biological Interactions, 167(1):63 – 70, 2007. Cited on page4.

[14] C Subhendu. Structural enzymology of human senescence marker protein 30 (SMP30): Insights into the gluconolactonase mechanism and role of metal ions. PhD thesis, University of Delaware, 2009. Cited on pages4,5, and7.

[15] M A Hicks. Analysis of the Nucleophilic Attack 6-bladed beta-Propeller Superfamily Provides Insight into the Evolution of Function in Strictosidine Synthase-like Proteins. PhD thesis, University of California, San Francisco, 2011. Cited on page5.

[16] Subhendu Chakraborti and Brian J Bahnson. Crystal Structure of Human Senescence Marker Protein 30: Insights Linking Structural, Enzymatic, and Physiological Func- tions,. Biochemistry, 2010. Cited on pages5,6, and7.

[17] Tatyana Belinskaya, Nagarajan Pattabiraman, Robert diTargiani, Moonsuk Choi, and Ashima Saxena. Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2012. Cited on pages5,6, and7.

[18] Yoshitaka Kondo, Akihito Ishigami, Sachiho Kubo, Setsuko Handa, Keiko Gomi, Kozo Hirokawa, Naoki Kajiyama, Tsuyoshi Chiba, Kentaro Shimokado, and Naoki Maruyama. Senescence marker protein-30 is a unique enzyme that hydrolyzes diisopropyl phosphoroﬂuoridate in the liver. {FEBS} Letters, 570(13):57 – 62, 2004. Cited on page6.

[19] Marc-Michael Blum, Frank Lhr, Andre Richardt, Heinz Rterjans, and Julian C.-H. Chen. Binding of a designed substrate analogue to diisopropyl ﬂuorophosphatase: 44 implications for the phosphotriesterase mechanism. Journal of the American Chemical Society, 128(39):12750–12757, 2006. PMID: 17002369. Cited on pages7 and8.

[20] Marc-Michael Blum, Marat Mustyakimov, Heinz Rüterjans, Kai Kehe, Benno P Schoenborn, Paul Langan, and Julian C-H Chen. Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and x-ray diffraction refinement. Proceedings of the National Academy of Sciences, 2009. Cited on pages7 and8.

[21] A K Katz, J P Glusker, and G D Markham. Deprotonation of water in the presence of carboxylate and magnesium ions. The Journal of Physical . . . , 1998. Cited on page8.

[22] Ann Babtie, Nobuhiko Tokuriki, and Florian Hollfelder. What makes an enzyme promiscuous? Current Opinion in Chemical Biology, 14(2):200 – 207, 2010. Bio- catalysis and Biotransformation/Bioinorganic Chemistry. Cited on pages8 and9.

[23] Kin-Yiu Wong and Jiali Gao. The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined qm/mm simulations. Biochemistry, 46(46):13352– 13369, 2007. PMID: 17966992. Cited on pages 10 and 11.

[24] Fernanda Ely, Kieran S. Hadler, Lawrence R. Gahan, Luke W. Guddat, David L. Ollis, and Gerhard Schenk. The organophosphate-degrading enzyme from agrobac- terium radiobacter displays mechanistic ﬂexibility for catalysis. Biochemical Journal, 432(3):565–573, 2010. Cited on pages 10 and 12.

[25] G A Omburo, J M Kuo, L S Mullins, and F M Raushel. Characterization of the zinc binding site of bacterial phosphotriesterase. Journal of Biological Chemistry, 267(19):13278–83, 1992. Cited on page 10.

[26] Suk-Bong Hong and Frank M. Raushel. Metalsubstrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase. Biochemistry, 35(33):10904–10912, 1996. PMID: 8718883. Cited on page 10.

[27] Fernanda Ely, KieranS. Hadler, Nataa Miti, LawrenceR. Gahan, DavidL. Ollis, NicholasM. Plugis, MarieT. Russo, JamesA. Larrabee, and Gerhard Schenk. Electronic and geometric structures of the organophosphate-degrading enzyme from agrobac- terium radiobacter (opda). JBIC Journal of Biological Inorganic Chemistry, 16(5):777– 787, 2011. Cited on pages 10, 11, and 13.

[28] Andrew N. Bigley and Frank M. Raushel. Catalytic mechanisms for phospho- triesterases. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1834(1):443 – 453, 2013. Special Section: Chemistry and mechanism of phosphatases, diesterases and triesterases. Cited on pages 10, 11, and 13.

[29] Cynthia R. Samples, Frank M. Raushel, and Victoria J. DeRose. Activation of the binuclear metal center through formation of phosphotriesteraseinhibitor complexes. Biochemistry, 46(11):3435–3442, 2007. PMID: 17315951. Cited on pages 10 and 14.

[30] George A. Omburo, Leisha S. Mullins, and Frank M. Raushel. Structural characterization of the divalent cation sites of bacterial phosphotriesterase by cadmium-113 nmr

45 spectroscopy. Biochemistry, 32(35):9148–9155, 1993. PMID: 8396425. Cited on pages 10, 14, and 15.

[31] Matthew M. Benning, Hyunbo Shim, Frank M. Raushel, and Hazel M. Holden. High resolution x-ray structures of diﬀerent metal-substituted forms of phosphotriesterase from pseudomonas diminuta,. Biochemistry, 40(9):2712–2722, 2001. PMID: 11258882. Cited on pages 10 and 14.

[32] Nicolai Lehnert, Serena DeBeer George, and Edward I. Solomon. Recent advances in bioinorganic spectroscopy. Current Opinion in Chemical Biology, 5(2):176 – 187, 2001. Cited on page 11.

[33] Martin L Kirk and Katrina Peariso. Recent applications of {MCD} spectroscopy to metalloenzymes. Current Opinion in Chemical Biology, 7(2):220 – 227, 2003. Cited on page 11.

[34] Jonathan McMaster and Vasily S Oganesyan. Magnetic circular dichroism spectroscopy as a probe of the structures of the metal sites in metalloproteins. Current Opinion in Structural Biology, 20(5):615 – 622, 2010. Cited on pages 11 and 13.

[35] Julien J.H. Cotelesage, M. Jake Pushie, Pawel Grochulski, Ingrid J. Pickering, and Graham N. George. Metalloprotein active site structure determination: Synergy between x-ray absorption spectroscopy and x-ray crystallography. Journal of Inorganic Biochemistry, 115(0):127 – 137, 2012. Special Issue in Memory of Hans Freeman. Cited on page 13.

[36] Yonglin Hu, Salem Faham, Roopali Roy, Michael W.W Adams, and Douglas C Rees. Formaldehyde ferredoxin oxidoreductase from pyrococcus furiosus: the 1.85 resolution crystal structure and its mechanistic implications. Journal of Molecular Biology, 286(3):899 – 914, 1999. Cited on page 14.

[37] Cynthia R. Samples, Timothy Howard, Frank M. Raushel, and Victoria J. DeRose. Protonation of the binuclear metal center within the active site of phosphotriesterase. Biochemistry, 44(33):11005–11013, 2005. PMID: 16101284. Cited on page 14.

[38] Richard Cammack. Electron paramagnetic resonance spectroscopy of metalloproteins. In Christopher Jones, Barbara Mulloy, and AdrianH. Thomas, editors, Spectroscopic Methods and Analyses, volume 17 of Methods in Molecular Biology, pages 327–344. Humana Press, 1993. Cited on page 14.

[39] Hongyan Li and Hongzhe Sun. Nmr studies of metalloproteins. In Guang Zhu, editor, NMR of Proteins and Small Biomolecules, volume 326 of Topics in Current Chemistry, pages 69–98. Springer Berlin Heidelberg, 2012. Cited on page 14.

[40] Yang Wang and Harold E. Van Wart. [14] raman and resonance raman spectroscopy. In James F. Riordan and Bert L. Vallee, editors, Metallobiochemistry Part C: Spectro- scopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins, volume 226 of Methods in Enzymology, pages 319 – 373. Academic Press, 1993. Cited on page 15.

46 [41] C.H. Evans. Biochemistry of the lanthanides. Biochemistry of the elements. Plenum Press, 1990. Cited on pages 15 and 30.

[42] Ane K. di Gennaro, Leonid Gurevich, Esben Skovsen, Michael T. Overgaard, and Peter Fojan. Study of the tryptophan-terbium fret pair coupled to silver nanoprisms for biosensing applications. Phys. Chem. Chem. Phys., 15:8838–8844, 2013. Cited on pages 15 and 16.

[43] J. Susanne Becker, Ryszard Lobinski, and J. Sabine Becker. Metal imaging in non- denaturating 2d electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (la-icp-ms) for the detection of metalloproteins. Metallomics, 1:312–316, 2009. Cited on page 16.

[44] E.G. Erds, C.R. Debay, and M.P. Westerman. Arylesterases in blood: Eﬀect of calcium and inhibitors. Biochemical Pharmacology, 5(3):173 – 186, 1960. Cited on page 17.

[45] M.Carmen Gonzalvo, Fernando Gil, Antonio F. Hernndez, Enrique Villanueva, and Antonio Pla. Inhibition of paraoxonase activity in human liver microsomes by exposure to edta, metals and mercurials. Chemico-Biological Interactions, 105(3):169 – 179, 1997. Cited on page 17.

[46] Chung-Liang Kuo and Bert N. La Du. Calcium binding by human and rabbit serum paraoxonases: Structural stability and enzymatic activity. Drug Metabolism and Dis- position, 26(7):653–660, 1998. Cited on page 17.

[47] S. Billecke, D. Draganov, R. Counsell, P. Stetson, C. Watson, C. Hsu, and B. N. La Du. Human serum paraoxonase (pon1) isozymes q and r hydrolyze lactones and cyclic carbonate esters. Drug Metabolism and Disposition, 28(11):1335–1342, 2000. Cited on page 17.

[48] Sivaramakrishnan Muthukrishnan, Vivekanand S. Shete, Toby T. Sanan, Shubham Vyas, Shameema Oottikkal, Lauren M. Porter, Thomas J. Magliery, and Christo- pher M. Hadad. Mechanistic insights into the hydrolysis of organophosphorus compounds by paraoxonase-1: exploring the limits of substrate tolerance in a promiscuous enzyme. Journal of Physical Organic Chemistry, 25(12):1247–1260, 2012. Cited on pages 20 and 22.

[49] Marion M. Bradford. A rapid and sensitive method for the quantitation of micro- gram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(12):248 – 254, 1976. Cited on page 21.

[50] James E. Noble and Marc J.A. Bailey. Chapter 8 quantitation of protein. In Richard R. Burgess and Murray P. Deutscher, editors, Guide to Protein Puriﬁcation, 2nd Edition, volume 463 of Methods in Enzymology, pages 73 – 95. Academic Press, 2009. Cited on page 21.

[51] Tsaﬀrir Zor and Zvi Selinger. Linearization of the bradford protein assay increases its sensitivity: Theoretical and experimental studies. Analytical Biochemistry, 236(2):302 – 308, 1996. Cited on page 21.

47 [52] J. F.; de Moreno, M. R.; Smith and R. V. Smith. Mechanism studies of coomassie blue and silver staining of proteins. J. Pharm. Sci., 75(9):907–11, 1986. Cited on page 21.

[53] Tamara C. Otto, Christina K. Harsch, David T. Yeung, Thomas J. Magliery, Dou- glas M. Cerasoli, and David E. Lenz. Dramatic diﬀerences in organophosphorus hydrolase activity between human and chimeric recombinant mammalian paraoxonase-1 enzymes. Biochemistry, 48(43):10416–10422, 2009. PMID: 19764813. Cited on page 22.

[54] Richard C Stevens, Stephanie M Suzuki, Toby B Cole, Sarah S Park, Rebecca J Richter, and Clement E Furlong. Engineered recombinant human paraoxonase 1 (rHuPON1) puriﬁed from Escherichia coli protects against organophosphate poisoning. Proceedings of the National Academy of Sciences, 2008. Cited on page 22.

[55] S Billecke, D Draganov, R Counsell, P Stetson, C Watson, C Hsu, and B N La Du. Human Serum Paraoxonase (pon1) Isozymes Q and R Hydrolyze Lactones and Cyclic Carbonate Esters. Drug Metabolism and Disposition, 2000. Cited on page 22.

[56] Olga Khersonsky and Dan S. Tawﬁk. Chromogenic and ﬂuorogenic assays for the lactonase activity of serum paraoxonases. ChemBioChem, 7(1):49–53, 2006. Cited on pages 22 and 23.

[57] Jose Ceron, Fernando Tecles, and Asta Tvarijonaviciute. Serum paraoxonase 1 (pon1) measurement: an update. BMC Veterinary Research, 10(1):74, 2014. Cited on page 23.

[58] Ronan Gilson and Marcus C. Durrant. Estimation of the pka values of water ligands in transition metal complexes using density functional theory with polarized continuum model solvent corrections. Dalton Trans., pages 10223–10230, 2009. Cited on page 34.

[59] Sandra E. Rodriguez-Cruz, Rebecca A. Jockusch, and Evan R. Williams. Binding energies of hexahydrated alkaline earth metal ions, m2+(h2o)6, m = mg, ca, sr, ba: evidence of isomeric structures for magnesium. Journal of the American Chemical Society, 121(9):1986–1987, 1999. PMID: 16429613. Cited on page 34.

[60] Lothar Helm and Andr E. Merbach. Inorganic and bioinorganic solvent exchange mechanisms. Chemical Reviews, 105(6):1923–1960, 2005. PMID: 15941206. Cited on pages 34, 36, 37, 38, 40, and 41.

[61] J. E. GORMAN and F. M. CLYDESDALE. Thermodynamic and kinetic stability constants of selected carboxylic acids and iron. Journal of Food Science, 49(2):500– 503, 1984. Cited on page 35.

[62] H Irving and R J P Williams. 637. The stability of transition-metal complexes. J. Chem. Soc., 1953. Cited on page 36.

[63] Olga Khersonsky and Dan S Tawﬁk. The histidine 115-histidine 134 dyad mediates the lactonase activity of mammalian serum paraoxonases. Journal of Biological Chemistry, 2006. Cited on page 41.

48 [64] Marc-Michael Blum, Christopher M Timperley, Gareth R Williams, Horst Thiermann, and Franz Worek. Inhibitory Potency against Human Acetylcholinesterase and Enzy- matic Hydrolysis of Fluorogenic Nerve Agent Mimics by Human Paraoxonase 1 and Squid Diisopropyl Fluorophosphatase. Biochemistry, 2008. Cited on pages 41 and 42.

[65] Xin Hu, Xiaohui Jiang, David E Lenz, Douglas M Cerasoli, and Anders Wallqvist. In silico analyses of substrate interactions with human serum paraoxonase 1. Proteins: Structure, Function, and Bioinformatics, 2009. Cited on page 41.

49 Appendix A pH Dependent PON1 Activity

Table A.1: Speciﬁc Activities of metal-substituted PON1s at pH 6.0, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 20±9 1±2 1±0 1±1 none 8±5 0±0 0±0 1±1 Ca2+ 9±3 0±0 0±0 1±0 Co2+ 6±3 0±0 0±0 1±0 Cu2+ 6±4 1±0 0±0 1±1 Eu3+ 6±3 0±0 0±0 1±1 Fe2+/Fe3+ 5±5 1±0 0±0 1±0 Mg2+ 8±5 0±0 0±0 1±1 Mn2+ 8±5 0±0 0±0 1±1 Ni2+ 5±3 0±0 0±0 1±1 Sr2+ 10±3 0±0 0±0 1±0 Zn2+ 8±5 0±0 0±0 1±1

50 Table A.2: Speciﬁc Activities of metal-substituted PON1s at pH 6.5, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 40±10 2±1 40±7 0.8±0.6 none 4±1 0.2±0.05 0.04±0.005 0.7±0.6 Ca2+ 20±10 0.1±0.04 0.2±0.007 1±0.4 Co2+ 10±8 0.2±0.03 0.06±0.01 0.6±0.4 Cu2+ 0.9±3 0.3±0.06 0.05±0.02 0.7±0.6 Eu3+ 2±2 0.3±0.08 0.07±0.03 0.6±0.5 Fe2+/Fe3+ 3±4 0.2±0.2 0.005±0.08 0.6±0.2 Mg2+ 4±0.8 0.6±0.6 0.07±0.04 0.6±0.5 Mn2+ 10±10 0.3±0.08 0.06±0.01 0.7±0.5 Ni2+ 10±10 0.3±0.03 0.2±0.04 0.6±0.5 Sr2+ 2±2 0.3±0.02 1±0.06 1±0.4 Zn2+ 7±2 0.3±0.06 0.06±0.01 0.7±0.7

Table A.3: Speciﬁc Activities of metal-substituted PON1s at pH 7.0, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 40±2 4±0.2 40±0.5 50±2 none 1±0.5 0.02±0.02 0.1±0.008 6±9 Ca2+ 70±5 8±0.5 70±2 90±3 Co2+ 1±0.3 0.7±0.1 20±3 2±0.4 Cu2+ 10±7 0.9±0.1 10±0.7 8±0.2 Eu3+ 0.4±0.2 0.3±0.03 2±0.6 1±0.5 Fe2+/Fe3+ 10±2 2±0.4 10±0.7 9±0.8 Mg2+ 8±2 0.3±0.04 30±20 10±0.5 Mn2+ 20±3 1±0.04 40±5 100±10 Ni2+ 2±0.6 0.07±0.01 1±0.04 7±6 Sr2+ 10±2 1±0.04 40±3 100±20 Zn2+ 0.4±0.1 0.1±0.002 20±0.2 6±8

51 Table A.4: Speciﬁc Activities of metal-substituted PON1s at pH 7.5, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 30±7 9±1 80±8 200±20 none 0.4±0.2 0.03±0.008 0.09±0.01 1±0.2 Ca2+ 80±10 20±3 100±10 300±30 Co2+ 3±0.2 0.2±0.01 9±1 30±4 Cu2+ 30±1 3±0.06 40±5 90±10 Eu3+ 3±0.3 0.7±0.02 5±1 20±3 Fe2+/Fe3+ 80±9 7±0.4 80±5 200±30 Mg2+ 3±0.1 0.6±0.04 90±20 80±70 Mn2+ 60±1 3±0.2 70±10 300±100 Ni2+ 0.4±0.2 0.07±0.01 1±0.2 2±0.1 Sr2+ 30±2 2±0.03 80±20 200±30 Zn2+ 0.9±0.09 0.1±0.01 10±0.8 4±0.2

Table A.5: Speciﬁc Activities of metal-substituted PON1s at pH 8.0, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 90±10 20±1 300±6 300±50 none 10±7 0.05±0.004 0.09±0.01 80±10 Ca2+ 200±8 30±0.7 300±40 600±90 Co2+ 6±0.4 0.2±0.006 20±2 90±10 Cu2+ 8±2 2±0.09 40±3 60±10 Eu3+ 80±10 3±0.2 70±8 40±7 Fe2+/Fe3+ 100±8 2±0.7 200±20 300±30 Mg2+ 200±9 0.7±0.05 200±30 400±70 Mn2+ 70±7 2±0.02 200±20 300±80 Ni2+ 1±1 0.2±0.03 2±0.3 30±7 Sr2+ 80±5 3±0.01 80±3 500±20 Zn2+ 0.6±0.6 4±0.2 30±0.1 30±7

52 Table A.6: Speciﬁc Activities of metal-substituted PON1s at pH 8.5, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 200±20 7±0.4 60±2 200±100 none 30±2 0.05±0.02 0.07±0.009 100±4 Ca2+ 200±9 20±1 200±9 500±20 Co2+ 10±2 0.4±0.02 50±70 80±8 Cu2+ 30±2 10±0.8 50±6 200±3 Eu3+ 60±8 3±0.07 30±1 100±1 Fe2+/Fe3+ 80±6 0.8±0.3 100±10 300±20 Mg2+ 200±8 1±0.03 200±20 400±90 Mn2+ 30±1 2±0.1 100±20 400±40 Ni2+ 9±1 0.08±0.004 1±0.1 50±3 Sr2+ 40±0.7 3±0.09 80±20 500±40 Zn2+ 0.6±0.4 0.3±0.02 6±1 50±8

Table A.7: Speciﬁc Activities of metal-substituted PON1s at pH 9.0, unit: mM−1 min−1

PA Px TBBL DHC Original PON1 70±2 20±2 400±60 600±200 none 0.3±0.2 0.05±0.02 0.07±0.0007 3±2 Ca2+ 100±10 20±1 300±100 400±200 Co2+ 20±3 1±0.1 30±2 100±70 Cu2+ 90±2 10±1 100±40 400±200 Eu3+ 20±2 5±0.3 90±5 200±90 Fe2+/Fe3+ 50±5 -0.009±0.07 100±20 300±200 Mg2+ 8±0.8 0.7±0.09 100±50 300±100 Mn2+ 60±10 2±0.4 100±30 400±300 Ni2+ 3±0.3 0.1±0.004 10±2 50±30 Sr2+ 70±7 4±0.2 70±4 400±200 Zn2+ 90±4 3±0.2 50±10 400±200

53 54

Figure A.1: pH value dependent PON1 activity - Px 55

Figure A.2: pH value dependent PON1 activity - TBBL 56

Figure A.3: pH value dependent PON1 activity - PA 57

Figure A.4: pH value dependent PON1 activity - DHC Appendix B Jahn-Teller Distortion and Lability of Cu(II)

Jahn-Teller theorem is for the feature of distortion in the octahedral symmetry. The description is following

In any non-linear system, there exists a vibrational mode that removes the degeneracy of an orbitally degenerate state.

The reason for Jahn-Teller distortion happens is for reduce the degeneracy of electron states, 6 3 in order to stabilize the overall energy. For example, t2geg ground state conﬁguration. In 10 other words, the ’hole’ in the d conﬁguration can occupy either dz2 or dx2−y2 . In the case

of ’hole’ in dz2 , the dx2−y2 axis will be elongated for reduce the energy of dx2−y2 , which

simultaneously enhances the energy of dz2 . As the results, the state which has one electron

(dz2 orbital) become the anti-bonding with the highest orbital, and reduce the overall energy of complex. Jahn-Teller distortion is common in many hexa-coordinate copper(II) complexes. The four ligands in the equatorial plane will be shorten and the dz2 orbital will be elongated. In copper(II) complexes, the distortion axis jumps very rapidly. That is to say, all ligands will vibrate and switch between equatorial and axial in time. For distorted complex, the axial ligands bind to the metal loosely, which is highly labile for solvent exchange.