Accepted Manuscript

Title: Conversion of non-allosteric methylglyoxal synthaseinto a homotropic allosteric enzyme by C-terminal deletion

Author: Malihe Mohammadi Shekufeh Zareian KhosroKhajeh

PII: S1381-1177(14)00144-1DOI: http://dx.doi.org/doi:10.1016/j.molcatb.2014.04.022Reference: MOLCAB 2948

To appear in: Journal of Molecular Catalysis B: Enzymatic

Received date: 29-8-2013Revised date: 25-4-2014Accepted date: 30-4-2014

Please cite this article as: M. Mohammadi, S. Zareian, K. Khajeh, Conversionof non-allosteric methylglyoxal synthase into a homotropic allosteric enzymeby C-terminal deletion, Journal of Molecular Catalysis B: Enzymatic (2014),http://dx.doi.org/10.1016/j.molcatb.2014.04.022

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Conversion of non-allosteric methylglyoxal synthase into a homotropic allosteric enzyme by C-terminal deletion

Malihe Mohammadi1*, Shekufeh Zareian2* and Khosro Khajeh1†

1 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences, Zanjan, Iran

Running Title: Effect of C-terminal deletion on EMGS properties

* These authors contributed equally to this work.

†Corresponding author: Khosro Khajeh, Department of Biochemistry, Faculty of biological

Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran. Tel/Fax; +98-21-

82884717; E-mail: khajeh@modares.ac.ir

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Abbreviations:

EMGS: E.coli Methylglyoxal Synthase

TMGS: Thermus sp. GH5 Methylglyoxal Synthase

DHAP: Dihydroxyacetone Phosphate

Highlights

EMGS-∆C revealed homotropic cooperative behavior

EMGS-ΔC is more flexible and less stable compared to wild-type

EMGS C-terminal tail plays an important role in allosteric behavior

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Abstract

Our previous study revealed that the Hill coefficient of E. coli methylglyoxal synthase (EMGS)

is higher than what we have calculated for MGS from Thermus sp. GH5 (TMGS). Amino acid

sequence alignment of EMGS and TMGS shows that key residues of allosteric pathways in

EMGS exist in TMGS as well, except Arg150 which plays a crucial role in forming a salt bridge

with Asp20 in the neighboring subunit and consequently transfers the allosteric signal between

the subunits. To equalize allosteric pathway in EMGS with TMGS, ten amino acid residues,

containing Arg150, are omitted from the EMGS C-terminal tail. The resulting recombinant

enzyme (EMGS-∆C) surprisingly shows homotropic cooperative behavior in presence of

dihydroxyacetone phosphate. Structural studies and irreversible thermoinactivation data shows

EMGS-ΔC is not only more flexible but also less stable compared to wild-type EMGS. These

data suggest EMGS C-terminal tail may play an important role in allosteric behavior and stability

of wild-type EMGS and thus indicating that the homotropic cooperatvity is arisen by binding of

the substrate which pushes the pre-existing equilibrium between the relatively inactive (RI) and

relatively active (RA) conformations.

Keywords: E. coli methylglyoxal synthase; Allosteric pathway; Hill coefficient; Cooperativity.

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1. Introduction

Allostery, or a “distinct site”, is the regulation of enzyme activity between two separated sites [1-

3]. All allosteric proteins are either oligomeric or contain multiple interacting domains within

one polypeptide chain [4, 5], and display homotropic or heterotropic cooperativity in ligand

binding and a characteristically sigmoidal dependence of reaction velocity on substrate

concentration [5]. Two models have been advanced to explain the binding properties of allosteric

ligands [6, 7]. In contrary to the classical view, the allosteric perturbation does not necessarily

arise from binding a ligand but any type of alteration in the protein could commence allosteric

signals [5, 8-12]. However, there are a lot of oligomeric enzymes which have not cooperative

behavior and exhibit standard Michaelis-Menten kinetics [8, 13].

Methylglyoxal synthase (MGS, EC 4.2.3.3) catalyzes an elimination reaction which converts

dihydroxyacetone phosphate (DHAP) to methylglyoxal (MG) and phosphate [14, 15]. MGS is a

homohexameric enzyme, and phosphate inhibits it allosterically. This could suggest a role for

this enzyme under phosphate deficiency condition [16]. For the wild-type MGS in the presence

of phosphate, cooperativity emerges between active sites upon substrate binding [17]. Although,

this enzyme has been purified from different sources, but MGS from E. coli (EMGS) has been

studied the most. EMGS gene is composed of 459 bp which encodes a polypeptide of 152 amino

acids with a molecular mass of 17 kDa [18]. The three dimensional structure of EMGS has been

determined by Saadat et al [19]. Sequence alignment with MGS from several bacterial species

indicates the absolute conservation of several amino acid residues: Asp 20, Asp 71, Asp 91, Asp

101, Arg 107, His 19, His 98 and Lys 21 [20]. Site-directed mutagenesis study suggests that Asp

71, Asp 101 and His 98 are involved in the enzyme catalysis [20]. According to the reported

crystal structures two pathways have been proposed through which EMGS transmits the

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allosteric information. In the first pathway, formation of a salt bridge between Asp 20 and Arg

150 in the presence of phosphate passes information between the six adjacent subunits. In the

second putative mechanism, Pro 92, Arg 107, and Val 111 are employed in this conveyance [19].

Recently, a gene encoding MGS from Thermus sp. GH5 (TMGS) was cloned, expressed [21] and

protein structure was studied by X-ray crystallography (PDB code 2XW6) [A. Shahsavar et al.

Unpublished results]. The amino acid sequence comparison of TMGS and EMGS enzymes has

shown 66% similarity. Sequence analysis has revealed that amino acids 143–152 which form the

C-terminal helix in EMGS are not present in TMGS (Fig. 1). TMGS has a lower cooperativity in

presence of phosphate than EMGS [21], thus the absence of Arg 150 in TMGS, comparing to

EMGS, has caused a decrease in the cooperativity between the enzyme subunits [21, 22].

Construction of this pathway by addition of a ten-residual sequence at the carboxy terminus of

TMGS increased the cooperative function of the enzyme [23]. Here to investigate the importance

of the mentioned C-terminal sequence role in the salt bridge formation and transmitting the

allosteric signal, ten amino acid sequence containing crucial Arg for salt bridge formation, were

omitted from the C-terminus of EMGS. In this way we are trying to equalize this enzyme to

TMGS. The new enzyme was called EMGS-∆C. The apparent decrease in nH of EMGS-∆C was

expected, but the results show that the enzyme is behaving like a homotropic cooperative

enzyme in absence of phosphate, which means EMGS-∆C kinetic data are sigmoidal with respect

to substrate concentration. In the current study we are investigating the possible cause of

emergence of the new allosteric behavior in EMGS in consequence of this deletion. For this

reason thermal stability and changes in secondary and tertiary structures of these enzymes are

investigated by circular dichroism (CD) and fluorescence spectroscopy.

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2. Materials and Methods

2.1. Chemicals

T4-DNA ligase and restriction enzymes were purchased from Fermentas Life Science (Vilnius,

Lithuania). Oligonucleotides were synthesized by Macrogen Inc Company (Korea). Tryptone

and yeast extract were purchased from Liofilchem (Roseto degli Abruzzi, Italy).

Dihydroxyacetone phosphate was purchased from Sigma-Aldrich (USA). 2,4-

Dinitrophenylhydrazine and other chemicals were obtained from Merck (Darmstadt, Germany).

2.2. Cloning of E. coli MGS (EMGS)

Genomic DNA from Escherichia coli was prepared using DNA extraction kits. The EMGS gene

was amplified from genomic DNA by PCR using the following primers: forward (5′-

GGAATTCCATATGGAACTGACGACTCGCACTTTACC-3′) and reverse (5′-

CGCAAGCTTTTACTTCAGACGGTCCGCGAGATAAC-3′) to introduce the flanking NdeI

and HindIII restriction sites (the underlined bases specify the NdeI and Hind III restriction sites

in forward and reverse primers, respectively). The resulting fragment (459 bp) digested with

NdeI and HindIII and ligated into similarly digested pET-21a(+) vector using T4 DNA ligase.

Sequence integrity was confirmed by DNA sequencing.

2.3. Construction of EMGS-∆C plasmid

EMGS-∆C gene, (EMGS gene minus 30 bp at 3´-end) was constructed by using pET-21a(+)

plasmid containing the EMGS gene as a template. 0.08 µM forward (5´-

GGAATTCCATATGGAACTGACGACTCGCACTTTACC-3´) and reverse (5´-

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CGGAAGCTTTTAATCTGGGATCAGAATATCGACCGCG-3´) primers were used to amplify

EMGS-∆C, the other conditions are as explained in previous part. The resulting fragment (429

bp) digested with HindIII and NdeI and ligated into similarly digested pET-21a(+) vector using

T4 DNA ligase. Sequence integrity was confirmed by DNA sequencing.

2.4. Protein expression and purification

E. coli BL-21 cells harboring each of recombinant plasmids were grown in Luria–Bertani (LB)

medium supplemented with ampicillin (100 µg/mL) at 37 °C, 220 rpm. IPTG (1mM) was added

to the culture medium once the culture reached an optical density of 0.5 to 0.7 at 600 nm.

Subsequently, temperature was lowered from 37 to 30 °C suitable for production of adequate

amount of protein. After 19 hours, cells were harvested by centrifugation at 5000 rpm for 20 min

and resuspended in lysis buffer containing 2 mM imidazole, 100 mM NaCl and 50 mM Tris (pH

7.0). The suspension was subjected to sonic disruption and total lysate was centrifuged for 20

min at 12000 rpm at 4 °C. The supernatant was dialyzed against 20 mM Tris buffer, pH 8.0 to

apply onto a Q-Sepharose equilibrated with the same buffer. Proteins were eluted with a linear

gradient of NaCl (0-1 M) prepared in 20 mM Tris buffer (pH 8.0). The flow rate was set at 3

mL/min, and fractions containing MGS activity were collected. The purity of proteins was

confirmed by SDS-PAGE according to the method of Laemmli [24]. Protein concentration was

measured by Bradford method [25], using bovine serum albumin as standard.

2.5. Enzyme assay and kinetic characterization

The spectrophotometric assay of Hopper and Cooper [26] was used to determine the enzyme

activity. Briefly, 125 µL of 50 mM imidazole buffer (pH 7.0), 10 µL DHAP (15.625 mM) and 10

µL (16.8 ng) of the enzyme were incubated at 60 °C for 5 min. Then 0.1 mL of the mixture was

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added to 0.33 mL of 2,4-dinitro-phenyhydrazine reagent (0.1 % 2,4-dinitro-phenyhydrazine in 2

mM HCl) and followed by mixing with 0.9 mL of distilled water. After incubation in 30 °C for

15 min, 1.67 mL NaOH (10 % w/v) was added and the appeared purple color was measured at

550 nm after a further 15 min incubation. A molar extinction coefficient of 4.48 × 104 M-1 cm-1

was used to calculate the methyglyoxal concentration [16, 27].

Kinetic parameters measurements were carried out using different substrate concentrations (1-2.5

mM). Mixtures containing different concentrations of DHAP with no enzyme were used as

controls and each data point (initial velocity) was determined in triplicate. Steady-state kinetic

parameters in the presence and absence of phosphate were fitted to Michaelis-Menten equation

and were numerically analyzed by Lineweaver-Burk equation. Hill coefficient was calculated

from the fallowing equation:

Log [v/ (Vmax – v)] = nH log[S] – log (K´)

Where v and Vmax are velocity and maximal velocity of the enzyme, and nH is the Hill

coefficient. K´ is related to Km but also contains terms related to the effect of substrate occupancy

at one site on the substrate affinity of the other sites. According to this equation, the value of nH

can be calculated by plotting log [v/ (Vmax – v)] against log[S].

2.6. Intrinsic fluorescence and acrylamide quenching experiments

Tryptophan fluorescence of EMGS and EMGS-∆C was measured using a Perkin Elmer

luminescence spectrometer LS 55. Samples were excited at 280 nm and the emission was

recorded between 300 to 400 nm. All experiments were carried out at room temperature and

protein concentrations were 20 μM in 20 mM Tris buffer (pH 8.0). For quenching measurements,

different concentrations of acrylamide varying from 0 to 200 mM were obtained from a stock of

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acrylamide (1 M). After 5 min incubation with acrylamide, samples were excited at 280 nm and

the emission spectra were scanned between 300-500 nm. According to Stern-Volmer equation,

the fluorescence intensity at λmax of emission was analyzed:

Fο/F = 1 + Ksv [Q]

In this equation Fο and F are the fluorescence intensities at the emission Amax in the absence and

presence of quencher, [Q] is the quencher concentration, and Ksv is the quenching constant [28].

2.7. Circular dichroism measurements

Far-UV spectra (190-260 nm) were recorded on a Jasco spectropolarimeter J-715 (Tokyo, Japan)

using 1 mm path length quartz cell at the protein concentration of 0.2 mg/mL in 20 mM Tris

buffer (pH 8.0). Results are presented as molar ellipticity [θ] (deg cm2 dmol−1), based on a mean

amino acid residue weight of 110 for EMGS. The molar ellipticity [θ] was calculated from the

formula [θ]λ = (θ × 100MWR)/ (cl), where c is the protein concentration in mg/mL, l the light

path length in centimeters, and θ is the measured ellipticity in degrees at wavelength λ.

2.8. Irreversible thermoinactivation

Thermal inactivation of EMGS and EMGS-ΔC was investigated in 50 mM imidazole buffer (pH

7.0) at 60 and 65 °C. Periodically, after chilling on ice for 30 min, the residual activity of

enzymes were measured as described above. The untreated sample was used as a control (100%

activity).

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3. Results

3.1. Kinetic properties of EMGS-ΔC in comparison with wild-type EMGS

E. coli MGS (EMGS) and EMGS-ΔC genes were cloned, expressed and the enzymes purified on

a Q-Sepharose column at pH 8.0, as described in the methods section. These two enzyme

variants were dialyzed in Tris-HCl, pH 7.0 and then were used to determine the kinetic

parameters and Hill coefficients using Prism software version 5.04 (available at

www.graphpad.com). (Fig 2). The Hill coefficient and kcat/Km for the mutant variant in absence

of phosphate were 1.30 and 1.58, respectively, whereas these values for the wild-type were 1.0

and 8.43 (Table 1). These data indicate that the EMGS-ΔC variant is showing homotropic

cooperative behavior in absence of phosphate. Furthermore Hill coefficients of the mutant and

wild-type EMGS were determined in some different concentrations of phosphate (Table 2). Data

exhibit nH of the mutant enzyme in each concentration of phosphate, is decreased in comparison

with the native enzyme. Within ten residues deleted from EMGS’s C-terminus there is no

conserved residue or any directly involved residue in EMGS catalysis [18] (Fig. 1), but the C-

terminal α-helix makes interactions with the active site of the neighboring subunit [19, 20]. We

can conclude that probable loss of these interactions in the mutant enzyme have some effects on

the catalytic inefficiency. Also previously in the case of E. coli ornithine transcarbamoylase,

Dembowski et al reported that conferral of cooperative behavior to this enzyme was associated

with the reduction in its catalytic efficiency [29]. On the other hand the deleted sequence

contains an Arg (No. 150) which in the presence of phosphate forms a salt bridge with both Asp

20 and phosphate ion [19], so probably its deletion and lack of these interactions in EMGS-ΔC,

can be the cause of decreased Hill coefficient in presence of phosphate in comparison with native

enzyme (Table 2).

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3.2. Looser structure of EMGS-∆C is evident by fluorescence data

Further insights on structural changes induced by C-terminal deletion, arise from the differences

in fluorescence emission of mutant and wild type enzymes. The emission spectrum of a protein is

related to its aromatic residues, such as tryptophan and tyrosine, and it is strongly affected by the

microenvironment surrounding these fluorophores. Any structural alteration which changes the

position of these residues in a protein, can affect their emission spectrum as well. Therefore, it is

possible to indirectly follow the conformational changes of the protein through its intrinsic

fluorescence emission. Less structural compactness leads to decrease in the intensity of

fluorescence. As shown in Fig.3 A, a decrease in fluorescence intensity is observed upon ten

amino acid deletion from EMGS, so it can be proposed that C-terminal deletion has caused the

weakening of intersubunits interactions. But a more closely look into the mentioned C-terminal

sequence (143-YQRYLADRLK 152) shows that two tyrosine residues exist in this sequence. So

these two residues are absent in EMGS-ΔC and the decrease in fluorescence intensity may be the

effect of this absence. For measuring the structural flexibility and integrity of enzymes’ structure,

we performed fluorescence quenching assay using acrylamide [28]. The Stern-Volmer plots for

acrylamide quenching of wild-type EMGS and EMGS-ΔC are shown in Fig.3 B. The quenching

constants (Ksv values) for wild-type and EMGS-ΔC were calculated and found to be 1.67 and

2.16 (M-1), respectively. It is evident in the data that the EMGS-ΔC mutant is effectively

quenched by acrylamide. This could be indicative of more flexibility of EMGS-ΔC than wild-

type, which lets the tryptophan residues be readily accessible to acrylamide. This data confirms

fluorescence emission measurements.

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3.3. Circular dichroism spectra of EMGS and EMGS-ΔC

To evaluate the changes in the secondary structure of enzymes, far-UV CD measurements were

carried. The far-UV CD spectra of EMGS shows a slightly decrease in negative ellipticity upon

ten residue deletion which indicates a lesser secondary structure content in this mutant (Fig.3 C),

as we mentioned in the previous sections C-terminal residues are in the α-helix structure.

3.4. Deletion of C-terminal tail from EMGS lowered the thermal stability

Thermal inactivation of purified EMGS and EMGS-ΔC was performed in support of the

fluorescence spectroscopy data. The residual activity of enzymes after incubation at 60 °C

(optimum temperature of wild-type EMGS) [S. Zareian et al. Unpublished results] and 65 ºC was

measured to assess their thermal stability. As depicted in Fig.4 A, after heat treatment for 120

minutes at 60 °C, wild-type and EMGS-ΔC retained nearly 40% and 25% of their initial activity.

But after 60 minutes of incubation at 65 ºC, EMGS-ΔC loses its activity totally, whereas 15% of

initial activity of wild-type enzyme still remains (Fig.4 B). Taken together with fluorescence

data, it could be concluded that EMGS-ΔC is not only more flexible but also less stable than

wild-type EMGS.

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4. Discussion

The vital role of allostery is evident by various processes in the cells which are regulated by

allosteric proteins including metabolic and signal transduction pathways [3, 4, 30, 31,] Allostery

is cooperative event, positive or negative; up- or down- regulating protein functions [32]. All

dynamic (non-fibrous) proteins are potentially allosteric [32, 33]. Thousands of studies over

years have provided two types of examples of allosteric proteins; those that lose their

cooperativity [29, 31] and those which gain cooperative behavior upon changes induced by

structural and/or dynamic alterations [5, 8-12]. In the present study, it is demonstrate that a ten

residue deletion from C terminus of an oligomeric enzyme could be sufficient to trigger an

allosteric pathway in the enzyme.

There is an idea that proteins should be considered as a dynamic ensemble of conformational

states [34]. Allostery derives from populations and redistribution of the conformational ensemble

[33-36]. In the case of non-allosteric proteins, they are probably to be allosteric if modified by

any structural perturbation (i. e., mutations) or proper ligands. Mutations or ligands can facilitate

the transition of the protein from one conformation to another [2, 33, 37, 38]. This is so-called

‘‘new view’’ of allosteric transitions, that often referred to as the ‘‘population-shift’’ model [3,

33, 39, 40]. The model emphasizes that the activated conformation (i.e., the dominant

conformation after the allosteric transition) has a non-negligible population prior to activation,

and that the allosteric event (i.e., homotropic or heterotropic binding) shifts the pre-existing

equilibrium between the low- and high-activity conformations toward the latter which is now

relatively more stable [38-44]. Therefore, the shift in the pre-existing ensemble can lead to

different observed conformational dynamic and functional effects [40, 43].

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In the case of MGS, removal or disruption of some interactions in the interface of subunits

converts the active form of EMGS (form A) with the tightly bound subunits to a loosely bound

form which is relatively inactive (RI form) and results in a cooperative enzyme. It is proposed

that RI form of the enzyme is in equilibrium with the relatively active (RA) form and homotropic

cooperatvity is arisen by binding of the substrate which pushes this equilibrium towards RA form

[10, 22]. In reality, ligand binding destabilizes a conformation (RI form) favoring another

conformation (RA form) [4].

In summary, here for the first time we study the effect of C-terminal deletion on cooperative

behavior of EMGS and data exhibits that the enzyme is showing cooperative behavior in absence

of its allosteric ligand (phosphate). Fluorescence study and quenching assay using acrylamide

show that the overall structural compactness of EMGS-ΔC has decreased in comparison with the

wild-type. Also thermal inactivation data confirms that EMGS-ΔC is less stable compared to

wild-type EMGS. A key requirement for allostery is protein flexibility. Flexibility is a property

common to most proteins and links structure to function by allowing the communication between

very distant parts of a macromolecule [3, 4, 44]. Taken together our study is a good evidence for

the fact that enzymes are in a dynamic equilibrium between active and inactive conformation and

allosteric enzymes have such a potential structure which any modification in them can cause a

new allosteric behavior in response to their substrates or maybe new ligands, once they receive

the precondition for initiation of the allosteric pathway.

Nevertheless further evidence and more detailed structural studies (i.e., crystallographic studies)

are required to help to explain what interaction(s) are omitted or likely which new interaction(s)

between protein subunits are formed by ten residue deletion from EMGS. Furthermore systemic

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deletions and point mutations at the C-terminal tail of EMGS are needed to determine which

amino acid(s) in this region are directly responsible to create an allosteric enzyme.

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Acknowledgments:

We would like to thank the research council of Tarbiat Modares University for the financial

support during the course of this project.

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Shyamsunder, R. D. Young,Proc. Natl Acad. Sci. USA 82 (1985) 5000–5004.

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Figure legends

Fig.1. Amino acid sequence alignment of Thermus sp. GH5 MGS (TMGS) and E. coli enzyme

(EMGS) shows 66% similarity. TMGS do not possess the ten amino acids compared to EMGS in

both terminals.

Fig. 2. Michaelis-Menten curves of (●) EMGS and (■) EMGS-∆C as a function of DHAP and

phosphate concentration

Fig.3. (A) Fluorescence emission spectra of EMGS (―) and EMGS-∆C (----) at 280 nm

excitation wavelength. (B) Stern-Volmer plot of fluorescence quenching by acrylamide.

Fluorescence quenching at 25 ºC for (●) EMGS and (■) EMGS-∆C enzymes. (C) Far-UV CD

spectra of EMGS (―) and EMGS-∆C (----). The experiments were performed in triplicate.

Fig.4. Thermal stability of the (●) EMGS and (■) EMGS-∆C at (A) 60 °C and (B) 65 °C.

Experiments were performed at least in triplicate and the standard deviations were within ±5% of

the experimental values.

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Table 1. Kinetic parameters of EMGS and EMGS-ΔC.

a Km for EMGS-ΔC is K0.5

b nH for EMGS and EMGS-ΔC are in absence of phosphate.

Enzyme Km (mM)a kcat (s-1) kcat/Km nHb

EMGS 2.74 ± 0.5 23.12 ± 0.2 8.43 1.0 ± 0.08

EMGS-ΔC 1.35 ± 0.5 2. 14 ± 0.5 1.58 1.30 ± 0.07

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Table 2. Hill coefficients of EMGS and EMGS-ΔC in different concentrations of phosphate.

Values are the averages of three experiments and standard errors are less than 8%.

[P] concentration 0 mM 0.2 mM 0.5 mM 1 mM

nH of EMGS 1±0.08 1.37±0.09 1.46±0.08 1.72±0.12

nH of EMGS-∆C 1.30±0.07 1.27±0.07 1.32±0.06 1.5±0.09

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Fig. 1.

Figure 1

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Fig. 2.

Figure 2

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Fig. 3.

Figure 3

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Fig. 4.

Figure 4