SharathBalakrishnaandAsmitaPrabhunedownloads.hindawi.com/journals/tswj/2014/216270.pdf · respective reaction was determined at , , , and C. Arrhenius plot was used to calculate the

Research ArticleEffect of pH on the Hydrolytic Kinetics of Gamma-GlutamylTransferase from Bacillus subtilis

Sharath Balakrishna and Asmita Prabhune

Division of Biochemical Sciences Room No 1846 National Chemical Laboratory Dr Homi Bhabha Road Pune 411008 India

Correspondence should be addressed to Asmita Prabhune aaprabhunenclresin

Received 17 November 2013 Accepted 18 January 2014 Published 24 February 2014

Academic Editors D Benke J C Gomez-Fernandez and A Surguchov

Copyright copy 2014 S Balakrishna and A Prabhune This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The effect of pH on the steady state kinetics of gamma-glutamyl transferase (GGT) from Bacillus subtilis was examined usingglutamyl-(3-carboxyl)-4-nitroanilide as the chromogenic reporter substrate The enzyme was active in the pH range 70ndash110 withthe optimum activity at pH 110 We noticed a pH dependent transformation in the nature of substrate consumption kineticsThe substrate saturation curves were hyperbolic in the pH range 70ndash90 but changed into sigmoid form at pH 100 and 110 Hillrsquoscoefficients were gt1 We also analysed the effect of pH on the structure of the enzymeThe circular dichroism spectra of the enzymesample at pH 90 and 110 were coincidental in both far and near UV regions indicating conservation of the secondary and tertiarystructures respectivelyThemolecular weight of the enzyme sample was the same in both pH 70 and 110 indicating conservation ofthe quaternary structure These results show that the kinetic transformation does not involve significant conformational changesCooperative binding of multiple substrate molecules may not be the basis for the sigmoid kinetics as only one substrate bindingsite has been noticed in the reported crystal structures of B subtilis GGT

1 Introduction

Gamma-glutamyl transferases (GGT) (EC2232) are a fam-ily of highly conserved enzymes occurring in archaebacteriaeubacteria fungi protozoa nematodes plants andmammals[1] They catalyse the exogenous removal of the terminal 120574-glutamyl moiety from amides or peptides and its subsequenttransfer towater (hydrolysis) or an acceptor amine (transpep-tidation) or a second substrate molecule (autotransfer) Theacceptor amine can be a peptide or an amino acid with a freeamino group The three reactions are represented below

120574GluCONHR +H2Olarrrarr Glu + RNH

2

(Hydrolysis)

120574GluCONHR1 + R2CONH2larrrarr 120574GluCOR2 + R1NH

2

(Transpeptidation)

2120574GluCONHRlarrrarr 120574Glu-GluCONHR + RNH2

(Autotranspeptidation)(1)

Mammalian GGTs are involved in xenobiotic detoxifica-tion [2] and homeostasis of glutathione [3] Human GGTplays a role in pathologies like metastasis [4] and drugresistance of malignant cells [5] cardiovascular diseases [6]inflammation [7] and diabetes [8] and neurodegenerativediseases [9] Plant GGTs are speculated to participate inthe synthesis of flavour compounds [10] There are indica-tions that Bacillus subtilis GGT catalyses the degradationof capsular 120574-polyglutamic acid [11] while its homologuein Bacillus anthracis (capD) mediates covalent anchorageof capsular 120574-poly-D-glutamic acid to the cell wall [12] InHelicobacter pylori and Neisseria meningitidis GGT helpsthe bacterium to colonize the intestinal epithelium [13] andthe brain [14] respectively So far no unifying function hasbeen found to explain the strong conservation across thephylogenetic treeWe are interested in the structure-functionrelationships of GGT We have crystallised B subtilis GGTand determined its X-ray crystallographic structure [15]In the present study we explore the effect of pH on thesteady state kinetics of B subtilis GGT and show its uniqueproperties

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 216270 6 pageshttpdxdoiorg1011552014216270

2 The Scientific World Journal

2 Materials and Methods

21Chemicals Glutamyl-(3-carboxyl)-4-nitroaniline ammo-nium salt was obtained from Fluka (Switzerland) All otherreagents used were from Sigma Chemicals (USA)

22 Cloning and Expression of B subtilis GGT TheGGTORFwas amplified from the genomicDNAofB subtilis strainwiththe primers 51015840ACG CGG CCA TGG TAA AGC CGC CCAAAA GCT31015840 (forward) and 31015840GTT TTT CTC GAG TTTACG TTT TAA ATT GCC GAT51015840 (reverse) The primersappend 51015840 and 31015840 termini of the amplicon with flanks bearingrecognition sites for the restriction enzymes NcoI and XhoIrespectively The amplicon was produced by 25 cycles of TaqDNA polymerase catalysed polymerase chain reaction Eachthermal cycle comprised of 30 s denaturation at 94∘C 30 sannealing at 50∘C and 90 s extension at 72∘C Both ampliconand pET26b (Novagen) plasmid DNA were digested withNcoI and XhoI and then ligated to generate pET28a-GGTconstruct The construct substitutes the N-terminal signalpeptide with E coli specific equivalent and appends a C-terminal hexahistidine tag

The construct was transformed into E coli expres-sion strain BL21 (Stratagene) For protein productionthe cells were grown in LB medium supplemented with30 120583gmL kanamycin When OD

600reached 05 the culture

was induced by adding isopropyl-b-D-thiogalactopyranoside(IPTG) to a final concentration of 1mM Thereafter theincubation was continued at 16∘C overnight The cells werepelleted by centrifugation at 6000 rpm and stored at minus20∘Ctill further use

23 Purification of GGT IPTG induced cell pellet wassuspended in 20mM sodium phosphate buffer pH 70 and500mM NaCl (buffer A) containing 20mM imidazole Thecell suspension was homogenised by two cycles of extrusionthrough French press at 1500 psi The homogenate wasclarified by centrifugation at 15000 rpm for 1 hr and filteredthrough 045 120583 membrane The filtrate was loaded onto1mL HisTrap column (Amersham Biosciences) containingprecharged high performance nickel sepharose preequili-brated with buffer A and washed with the same buffer tillOD280

of the effluent reached the baselineThe bound proteinwas eluted with a linear gradient of 20ndash500mM imidazolecontained in the binding buffer The fractions correspondingto the single major peak were pooled and loaded ontoHi-Load Superdex S200 (Amersham Biosciences) columnpreequilibrated with 100mM tris HCl pH 75 and 100mMNaCl Pure GGT eluted as a single prominent peak andwas confirmed by activity test and SDS-PAGE analysis Theprotein was concentrated to 10mgmL over a 10 kDa cutoffmembrane (Pall) and preserved at minus20∘C

24 Enzyme Assay The enzyme was assayed by modifiedmethod of Suzuki et al [16] Briefly 04 120583g of enzyme wasincubated at 30∘C for 5mins with the ammonium salt ofglutamyl-(3-carboxyl)-4-nitroaniline in 100mM buffer in a

final volume of 100 120583L Buffers used were Tricine for pH 70ndash90 and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)for pH 100ndash110 The reaction was arrested with 900 120583L of2M acetic acid and the absorbance measured at 410 nmSuitable blanks were used to correct for absorbance due tothe substrate One unit of enzymewas defined as 120583moles of 3-carboxyl-4-nitroaniline formed in one minuteThe assay waslinear with respect to time of incubation

25 Steady State Kinetics Substrate saturation plots wereprepared by measuring the reaction rates in a range ofsubstrate concentrations Concentrations were chosen to givea relatively even distribution of the data points119870

119898and119881max

were determined by fitting the velocities toMichaelis-Mentenequation by nonlinear regression method using Origin61

V = 119881max[119878]

119870119898+ [119878]

(2)

Formeasurementsmade at pH 100 and 110 the velocitieswere fitted by nonlinear regression to Hillrsquos equation of theform

V = 119881max[119878]ℎ

11987005+ [119878]ℎ (3)

where V is the initial velocity 119881max is maximal velocity 119870119898

and 11987005

are the substrate concentrations for half maximalactivity and ℎ is the measure of cooperativity between 119899interacting sites

26 Circular Dichroism (CD)Measurement CD spectra werecollected on a Jasco J-715 spectropolarimeter Near and farUV CD spectra (200ndash250 nm) were collected with 01 and1mgmL of the enzyme placed in a path length of 01 and1 cm respectively The final spectrum was an average of 5accumulations measured at a rate of 100 nmmin at 1 nmresolution

27 Molecular Weight Estimation by Size Exclusion Chro-matography 1x 100 cm Sephacryl S 200 was equilibrated with150mM NaCl in 50mMNa-K phosphate buffer pH 70 Theelution volume of blue dextran was considered as the voidvolume of the packed column The column was calibratedby passing cytochrome C (12000Da) carbonic anhydrase(24000Da) bovine serum albumin (66000Da) alcoholdehydrogenase (15000Da) and beta-amylase (200000Da) Acalibration curvewas constructed by plotting logarithmof themolecular weight of the individual marker against the ratio ofthe void (vo) and respective elution volumes (ve) A sampleof B subtilis GGT was passed through the calibrated columnand its vevo ratio was used to calculate the molecular weightfrom the calibration curveThe columnwas then equilibratedwith 100mMCAPSbuffer pH 110 and 150mMNaCl andusedto estimate the molecular weight of B subtilisGGT as before

28 Reversibility Study A sample of the enzyme was incu-bated in 50mM CAPS buffer pH 110 and 150mM NaCl for

The Scientific World Journal 3

48 hrs at 6∘CThe sample was then cleaned by dialysis against100mM tris HCl buffer pH 75 and 100mMNaCl

29 Thermodynamic Analysis The value of 119896cat for therespective reaction was determined at 20 30 40 and 50∘CArrhenius plot was used to calculate the activation energyThe data were fitted by linear regression to the equation

ln 119896cat = minus119864119886

119877119879

+ ln119860 (4)

where 119864119886is the activation energy 119877 is the gas constant 119879 is

the absolute temperature and 119860 is the frequency of collisionThe slop of this plot is equal to minus119864

119886119877

Eyringrsquos plot was used for the determination of enthalpyand entropy of activation The data were fitted by linearregression to the equation

ln119896cat119879

= ln 119896119861ℎ

+

Δ119878Dagger

119877

minus

Δ119867Dagger

119877119879

(5)

where ℎ is Planckrsquos constant 119896119861is Boltzmannrsquos constant 119877 is

gas constant and 119879 is absolute temperature From the plotΔ119867Dagger was calculated from the slope (= minusΔ119867Dagger119877) and Δ119878Dagger

from the119910-axis intercept Free energy of activation (Δ119866Dagger)wascalculated from the equation

Δ119866Dagger= Δ119867

Daggerminus 119879Δ119878

Dagger (6)

3 Results

31 Enzyme Preparation B subtilis GGT is expressed assingle polypeptide precursor which undergoes posttransla-tional intramolecular and autocatalytic cleavage to producea heterodimer [17] The 2 polypeptide chains of the matureheterodimer weigh 45 and 22KDa (Supplemental Figure1 available online at httpdxdoiorg1011552014216270)Expression of the active enzyme was temperature depen-dent Induction of mid-log cultures at 30∘C resulted inthe formation of unprocessed precursor Fully processedheterodimeric enzyme was produced when the cultures wereinduced at 16∘C

32 119870119898

and pH Optimum The kinetics of GGT catal-ysed hydrolysis was studied with 120574-glutamyl-(3-carboxyl)-4-nitroaniline as the substrate The enzyme was active betweenpH 70 and 110 The shape of the pH titration curve and theoptimum condition depended on the amount of substrateused in the assay The optimum was at pH 90 when theassay used lower substrate concentration (2mM) And theoptimumwas at pH 110 when higher substrate concentration(64mM) was used (Figure 1) 119870

119898was found to be 259 plusmn

19 167 plusmn 18 and 269 plusmn 27mM at pH 75 90 and 110respectively It was earlier reported to be 100mM at pH 90[18] Thus pH has negligible effect on the substrate affinitybetween pH 70 and 110 Among GGTs the 119870

119898of B subtilis

GGT is unusually high The reported 119870119898

values of otherGGTs are mostly in micromolar concentrations 68120583M in Ecoli GGT [16] 12 120583M in H pylori GGT [19] 7120583M in humanGGT [20] 5 120583M in rat [21] 2mM in rabbit GGT [22] and16mM in porcine GGT [23]

0

005

01

015

02

025

09

14

19

24

29

7 8 9 10 11

(mic

rom

oles

min

)

pH

Figure 1 Effect of pH on B subtilis GGT catalysed hydrolysisStandard assay reactions were performed with either 2mM (I) or64 mM (e) 120574-Glutamyl-(3-carboxyl)-4-nitroaniline

33 Kinetic Transformation Between pH 70 and 90 thesubstrate saturation kinetics follows the standard Michaelis-Menten form producing a hyperbolic response However atpH 100 and 110 the substrate saturation curves were of sig-moid form (Figure 2) Standard deviation was higher (gt10)and adjusted coefficient of determination (1198772) was relativelylower when the data was fitted to hyperbolic curve Better fitwas obtained with Hillrsquos equation (see supplementary Table1 for details) The Hillrsquos coefficient (ℎ) was 16 plusmn 05 in pH100 and 21 plusmn 04 at pH 110 Enzyme samples incubatedat pH 110 for sim48 hours produced hyperbolic saturationcurves when assayed at pH 90This indicates that the kinetictransformation between hyperbolic and sigmoid forms isreversible and does not involve any permanent changes in theenzyme

34 Effect of pH on Structure The influence of pH on thestructure of the enzyme was analysed to determine if anychanges accompanied the kinetic transformation Poten-tial changes in the secondary and tertiary structures wereanalysed by CD spectroscopy Absorbance in the far UV(200ndash250 nm) is mostly due to backbone amide bonds andtherefore spectral changes in this region represent alterationsin the secondary structure [24] Aromatic residues (Trp Tyrand Phe) absorb most of the UV in the near region (250ndash300 nm) spectral changes in this region therefore are dueto alterations in the tertiary structure [24] CD spectra of Bsubtilis GGT were collected in both far and near UV regionsusing samples present in pH 70 and 110 buffers The spectraproduced by samples in pH 70 and 110 were coincidental inboth the UV regions (Supplemental Figure 2)

We then examined the effect of pH on the oligomericstate of the enzyme sample The native molecular weightof the sample was analysed by gel-filtration method Theelution profiles in both pH 70 and 110 were coincidentaland corresponded to a molecular weight of 66KDa on thecalibration curve (Supplemental Figure 3) Conservation of


0 20 40 60 80 100 120 140

0

50

100

150

200

250

300

Substrate (mM)

(mic

rom

oles

s)

(a)

0 20 40 60 80 100 120 140

0

50

100

150

200

Substrate (mM)

(mic

rom

oles

s)

(b)

Figure 2 Effect of pH on the nature of saturation curve for B subtilis GGT catalysed hydrolysis of 120574-Glutamyl-(3-carboxyl)-4-nitroaniline(a) and (b) represent substrate saturation curves at pH 90 and pH 110 respectively

Table 1 Effect of pH on the thermodynamic parameters for the hydrolysis of 120574-Glutamyl-(3-carboxyl)-4-nitroaniline

pHActivation energy

(E119886)

KJmol

Activation enthalpy(Δ119867Dagger)KJmol

Activation entropy(Δ119878Dagger)Jmol K

Free energy of activation(Δ119866Dagger)KJmol

75 239 216 minus1477 656110 279 254 minus1061 571

the molecular weight indicates that the sample does notundergo oligomerization at higher pH

35 Thermodynamics of Hydrolysis across the pH RangeThermal dependence of 119896cat was determined in the range 20ndash50∘C and used to prepare Arrhenius (Supplemental Figure4(a)) and Eyring (Supplemental Figure 4(b)) plots Theexperiments were performed in pH 75 and 110 The plotswere linear in the experimental range The thermodynamicparameters for the formation of the activated complex aregiven in Table 1 The activation enthalpies are positive asreactions involving bond breakage are endothermic Thenegative value ofΔ119878Dagger indicates that the reactantsmust assumeprecise conformation and approach each other at preciseangle to form the transition state and that the moleculesare constrained in the activated state The positive valuefor Δ119866Dagger was expected as the formation of transition stateis nonspontaneous Thus the B subtilis GGT catalysedhydrolytic reactions in pH 75 and 110 are more or lessthermodynamically similar except for a small improvementin Δ119878Daggerat pH 110

4 Discussion

The kinetics of GGT particularly that of the mammalianhomologues have been extensively studied because of itsphysiological significance Among microbial GGTs thekinetics is mostly studied with the homologue from Ecoli Our detailed analysis of the enzymatic kinetics of B

subtilisGGT reveals interesting features that have so far beenoverlooked Between pH 70 and 110 the 119881max of hydrolysisprogressively increased producing maximum activity at pH110 This was contrary to the previously reported pH profilewherein the hydrolysis followed a bell-shaped curve withthe maxima at pH 90 [18] Our analysis indicated that thereported optimum was an artifact as the pH profile wasconstructed not from 119881max values but from comparativeassays at fixed substrate concentration The reported assaysemployed merely 1mM concentration of the substrate as theamount is the standard for most GGT assays and moreimportantly the 119870

119898of B subtilis GGT was unknown at that

time The lowered rates at pH 100 and 110 relative to pH90 under conditions of low substrate concentration were aconsequence of kinetic transformation At lower substrateconcentrations sigmoid kinetics typically produces slowerrates than the corresponding hyperbolic form We were ableto recreate the bell-shaped curve by measuring the activitieswith 2mMsubstrate and contrast it withmeasurementsmadewith 64mMsubstrate (Figure 1)The progressively increasingresponse at higher substrate concentration indicates theinvolvement of a critical acidic group that contributes to thecatalysis in its deprotonated form

The pH dependent transformation of the kinetics ofsubstrate consumption from hyperbolic to sigmoid formis the most important observation in this study Fromliterature we realised that a similar transitionary behaviourwas observed previously in mammalian GGTs from human[25] and rabbit liver [22] However this aspect has notreceived much attention The transition from hyperbolic to


sigmoid form is sharp and occurs between pH 90 and 100The kinetic data was analyzed using Hillrsquos equation Thisis a modification of Michaelis-Menten equation to accountfor multiple interacting binding sites Hillrsquos coefficient (ℎ)represents cooperativity between multiple (119899) binding sitesHillrsquos coefficient for hydrolysis in pH 100 and pH 110 wasgt1 indicating positive cooperativity between the interactingsites The kinetic transformation is probably due to theinvolvement of a group with a p119870

119886of sim95 which upon

deprotonation favours sigmoid kinetics We need to exploreif the critical deprotonated acidic group responsible for themaximal activity and the kinetic transformation are from thesame amino acid residue

Sigmoid curve is generally a feature of enzymes withmultiple substrate binding sites with dissimilar affinitiesBinding of a substratemolecule at one of sites either improvesor diminishes the binding affinity of the other sites Multiplebinding sites often arise due to oligomeric organization ofthe protein Our size exclusion chromatography experimentsunder conditions of pH 75 and 110 gave same molecularweight thus excluding oligomerization as the potential basisfor sigmoid kinetics Furthermore CD spectroscopy dataindicates the absence of any major structural changes Theseresults show that sigmoid kinetics arises due to nonconfor-mational reasons

GGT employs nucleophilic substitution mechanism ofcatalysis with theN-terminal threonine of the smaller subunitserving as the catalytic nucleophile [26]The crystal structureof B subtilis GGT in complex with glutamate has beendetermined [27] The structure shows a glutamate residuebound at the catalytically competent active site These factsshow that there is only one active site in GGT includ-ing the B subtilis homologue The possibility of a secondsubstrate molecule binding to the acceptor amine bindingsite is ruled out as sigmoid kinetics was evident in thetranspeptidation reactions also (data to be published) Thecrystal structures of B subtilis GGT have been determinedboth in the native and substrate conjugated forms [27] Theenzyme has a kidney shape with a groove on one side ofthe molecule The active site where the glutamate moietyof the substrate binds occurs as a finger-shaped depressiontowards the middle of the groove The region immediatelyoutside of the active site does not show any large pocketsthat might bind to a glutamate residue Therefore it isunlikely that the sigmoid kinetics arises due to cooperativebinding of multiple substrate molecules Sigmoid kineticsdue to monomeric enzyme with single substrate bindingsites has been extensively studied inmammalian glucokinaseThe sigmoid behaviour in glucokinase arises because ofits occurrence in two interconvertible conformations withdissimilar affinities for the substrate [28] Preequilibriumkinetics and nuclear magnetic resonance studies are neces-sary to elucidate the origins of sigmoid kinetics in B subtilisGGT

In conclusion our study shows the drastic effect of pHon the hydrolytic kinetics of B subtilis GGT Our data rulesout changes in substrate specificity conformation catalyticmechanism and oligomeric state as the cause for the kinetictransformation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] N D Rawlings F R Morton and A J Barrett ldquoMEROPS thepeptidase databaserdquo Nucleic Acids Research vol 34 pp D270ndashD272 2006

[2] C A Hinchman H Matsumoto T W Simmons and N Balla-tori ldquoIntrahepatic conversion of a glutathione conjugate to itsmercapturic acid metabolism of 1-chloro-24-dinitrobenzenein isolated perfused rat and guinea pig liversrdquo The Journal ofBiological Chemistry vol 266 no 33 pp 22179ndash22185 1991

[3] A Meister ldquoOn the enzymology of amino acid tansportrdquoScience vol 180 no 4081 pp 33ndash39 1973

[4] M Benlloch A Ortega P Ferrer et al ldquoAcceleration of glu-tathione efflux and inhibition of 120574-glutamyltranspeptidase sen-sitize metastatic B16 melanoma cells to endothelium-inducedcytotoxicityrdquoThe Journal of Biological Chemistry vol 280 no 8pp 6950ndash6959 2005

[5] A Pompella V deTata A Paolicchi and F Zunino ldquoExpressionof 120574-glutamyltransferase in cancer cells and its significance indrug resistancerdquo Biochemical Pharmacology vol 71 no 3 pp231ndash238 2006

[6] Y Hashimoto A Futamura H Nakarai and K NakaharaldquoBlood pressure levels of serum lipids liver enzymes and bloodglucose by aldehyde dehydrogenase 2 and drinking habit inJapanesemenrdquoAtherosclerosis vol 158 no 2 pp 465ndash470 2001

[7] J Singh J Chander S Singh G Singh and C K Atalldquo120574-Glutamyl transpeptidase a novel biochemical marker ininflammationrdquo Biochemical Pharmacology vol 35 no 21 pp3753ndash3760 1986

[8] D H Lee M H Ha J H Kim et al ldquo120574-glutamyltransferase anddiabetesmdasha 4 year follow-up studyrdquo Diabetologia vol 46 no 3pp 359ndash364 2003

[9] J Sian D T Dexter A J Lees S Daniel P Jenner and C DMarsden ldquoGlutathione-related enzymes in brain in Parkinsonrsquosdiseaserdquo Annals of Neurology vol 36 no 3 pp 356ndash361 1994

[10] M N Martin and J P Slovin ldquoPurified 120574-glutamyl transpep-tidases from tomato exhibit high affinity for glutathione andglutathione S-conjugatesrdquo Plant Physiology vol 122 no 4 pp1417ndash1426 2000

[11] K Kimura L P Tran I Uchida and Y Itoh ldquoCharacterizationof Bacillus subtilis 120574-glutamyltransferase and its involvement inthe degradation of capsule poly-120574-glutamaterdquoMicrobiology vol150 no 12 pp 4115ndash4123 2004

[12] T Candela and A Fouet ldquoBacillus anthracisCapD belonging tothe 120574-glutamyltranspeptidase family is required for the covalentanchoring of capsule to peptidoglycanrdquoMolecularMicrobiologyvol 57 no 3 pp 717ndash726 2005

[13] C Chevalier J Thiberge R L Ferrero and A LabigneldquoEssential role of Helicobacter pylori 120574-glutamyltranspeptidasefor the colonization of the gastric mucosa of micerdquo MolecularMicrobiology vol 31 no 5 pp 1359ndash1372 1999

[14] H Takahashi K Hirose and H Watanabe ldquoNecessity ofmeningococcal 120574-glutamyl aminopeptidase for Neisseriameningitidis growth in rat cerebrospinal fluid (CSF) andCSF-like mediumrdquo Journal of Bacteriology vol 186 no 1 pp244ndash247 2004


[15] B Sharath A A Prabhune C G Suresh A J Wilkinson and JA Brannigan ldquoCrystal structure of 120574 glutamyl transferase fromBacillus subtilisrdquo Protein Data Bank Accession no 2v36

[16] H Suzuki H Kumagai and T Tochikura ldquo120574-Glutamyltran-speptidase from Escherichia coli K-12 purification and proper-tiesrdquo Journal of Bacteriology vol 168 no 3 pp 1325ndash1331 1986

[17] T Okada H Suzuki K Wada H Kumagai and K FukuyamaldquoCrystal structure of the 120574-glutamyltranspeptidase precursorprotein from Escherichia coli structural changes upon autocat-alytic processing and implications for the maturation mecha-nismrdquo The Journal of Biological Chemistry vol 282 no 4 pp2433ndash2439 2007

[18] HMinami H Suzuki andH Kumagai ldquoAmutant Bacillus sub-tilis 120574-glutamyltranspeptidase specialized in hydrolysis activityrdquoFEMS Microbiology Letters vol 224 no 2 pp 169ndash173 2003

[19] G Boanca A Sand T Okada et al ldquoAutoprocessing of Heli-cobacter pylori 120574-glutamyltranspeptidase leads to the formationof a threonine-threonine catalytic dyadrdquoThe Journal of Biologi-cal Chemistry vol 282 no 1 pp 534ndash541 2007

[20] Y Ikeda J Fujii M E Anderson N Taniguchi and A MeisterldquoInvolvement of Ser-451 and Ser-452 in the catalysis of human120574-glutamyl transpeptidaserdquoThe Journal of Biological Chemistryvol 270 no 38 pp 22223ndash22228 1995

[21] J S Elce andB Broxmeyer ldquo120574Glutamyltransferase of rat kidneySimultaneous assay of the hydrolysis and transfer reactions with(glutamate 14C)glutathionerdquoBiochemical Journal vol 153 no 2pp 223ndash232 1976

[22] D Bagrel C Petitclerc F Schiele and G Siest ldquoSome kineticproperties of 120574-glutamyltransferase from rabbi liverrdquo Biochim-ica et Biophysica Acta vol 658 no 2 pp 220ndash231 1981

[23] J W London L M Shaw D Fetterolf and D GarfinkelldquoDetermination of themechanism and kinetic constants for hogkidney 120574 glutamyltransferaserdquo Biochemical Journal vol 157 no3 pp 609ndash617 1976

[24] SM Kelly andN C Price ldquoThe use of circular dichroism in theinvestigation of protein structure and functionrdquoCurrent Proteinand Peptide Science vol 1 no 4 pp 349ndash384 2000

[25] C PetitClerc F Schiele D Bagrel A Mahassen and G SiestldquoKinetic properties of 120574-glutamyltransferase from human liverrdquoClinical Chemistry vol 26 no 12 pp 1688ndash1693 1980

[26] J W Keillor R Castonguay and C Lherbet ldquo120574-glutamyltranspeptidase substrate specificity and catalytic mechanismrdquoMethods in Enzymology vol 401 pp 449ndash467 2005

[27] K Wada M Irie H Suzuki and K Fukuyama ldquoCrystalstructure of the halotolerant 120574-glutamyltranspeptidase fromBacillus subtilis in complex with glutamate reveals a uniquearchitecture of the solvent-exposed catalytic pocketrdquo FEBSJournal vol 277 no 4 pp 1000ndash1009 2010

[28] M Larion and B GMiller ldquoHomotropic allosteric regulation inmonomeric mammalian glucokinaserdquo Archives of Biochemistryand Biophysics vol 519 no 2 pp 103ndash111 2012

Submit your manuscripts athttpwwwhindawicom

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Enzyme Research



Microbiology


2 Materials and Methods

21Chemicals Glutamyl-(3-carboxyl)-4-nitroaniline ammo-nium salt was obtained from Fluka (Switzerland) All otherreagents used were from Sigma Chemicals (USA)

22 Cloning and Expression of B subtilis GGT TheGGTORFwas amplified from the genomicDNAofB subtilis strainwiththe primers 51015840ACG CGG CCA TGG TAA AGC CGC CCAAAA GCT31015840 (forward) and 31015840GTT TTT CTC GAG TTTACG TTT TAA ATT GCC GAT51015840 (reverse) The primersappend 51015840 and 31015840 termini of the amplicon with flanks bearingrecognition sites for the restriction enzymes NcoI and XhoIrespectively The amplicon was produced by 25 cycles of TaqDNA polymerase catalysed polymerase chain reaction Eachthermal cycle comprised of 30 s denaturation at 94∘C 30 sannealing at 50∘C and 90 s extension at 72∘C Both ampliconand pET26b (Novagen) plasmid DNA were digested withNcoI and XhoI and then ligated to generate pET28a-GGTconstruct The construct substitutes the N-terminal signalpeptide with E coli specific equivalent and appends a C-terminal hexahistidine tag

The construct was transformed into E coli expres-sion strain BL21 (Stratagene) For protein productionthe cells were grown in LB medium supplemented with30 120583gmL kanamycin When OD

600reached 05 the culture

was induced by adding isopropyl-b-D-thiogalactopyranoside(IPTG) to a final concentration of 1mM Thereafter theincubation was continued at 16∘C overnight The cells werepelleted by centrifugation at 6000 rpm and stored at minus20∘Ctill further use

23 Purification of GGT IPTG induced cell pellet wassuspended in 20mM sodium phosphate buffer pH 70 and500mM NaCl (buffer A) containing 20mM imidazole Thecell suspension was homogenised by two cycles of extrusionthrough French press at 1500 psi The homogenate wasclarified by centrifugation at 15000 rpm for 1 hr and filteredthrough 045 120583 membrane The filtrate was loaded onto1mL HisTrap column (Amersham Biosciences) containingprecharged high performance nickel sepharose preequili-brated with buffer A and washed with the same buffer tillOD280

of the effluent reached the baselineThe bound proteinwas eluted with a linear gradient of 20ndash500mM imidazolecontained in the binding buffer The fractions correspondingto the single major peak were pooled and loaded ontoHi-Load Superdex S200 (Amersham Biosciences) columnpreequilibrated with 100mM tris HCl pH 75 and 100mMNaCl Pure GGT eluted as a single prominent peak andwas confirmed by activity test and SDS-PAGE analysis Theprotein was concentrated to 10mgmL over a 10 kDa cutoffmembrane (Pall) and preserved at minus20∘C

24 Enzyme Assay The enzyme was assayed by modifiedmethod of Suzuki et al [16] Briefly 04 120583g of enzyme wasincubated at 30∘C for 5mins with the ammonium salt ofglutamyl-(3-carboxyl)-4-nitroaniline in 100mM buffer in a

final volume of 100 120583L Buffers used were Tricine for pH 70ndash90 and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)for pH 100ndash110 The reaction was arrested with 900 120583L of2M acetic acid and the absorbance measured at 410 nmSuitable blanks were used to correct for absorbance due tothe substrate One unit of enzymewas defined as 120583moles of 3-carboxyl-4-nitroaniline formed in one minuteThe assay waslinear with respect to time of incubation

25 Steady State Kinetics Substrate saturation plots wereprepared by measuring the reaction rates in a range ofsubstrate concentrations Concentrations were chosen to givea relatively even distribution of the data points119870

119898and119881max

were determined by fitting the velocities toMichaelis-Mentenequation by nonlinear regression method using Origin61

V = 119881max[119878]

119870119898+ [119878]

(2)

Formeasurementsmade at pH 100 and 110 the velocitieswere fitted by nonlinear regression to Hillrsquos equation of theform

V = 119881max[119878]ℎ

11987005+ [119878]ℎ (3)

where V is the initial velocity 119881max is maximal velocity 119870119898

and 11987005

are the substrate concentrations for half maximalactivity and ℎ is the measure of cooperativity between 119899interacting sites

26 Circular Dichroism (CD)Measurement CD spectra werecollected on a Jasco J-715 spectropolarimeter Near and farUV CD spectra (200ndash250 nm) were collected with 01 and1mgmL of the enzyme placed in a path length of 01 and1 cm respectively The final spectrum was an average of 5accumulations measured at a rate of 100 nmmin at 1 nmresolution

27 Molecular Weight Estimation by Size Exclusion Chro-matography 1x 100 cm Sephacryl S 200 was equilibrated with150mM NaCl in 50mMNa-K phosphate buffer pH 70 Theelution volume of blue dextran was considered as the voidvolume of the packed column The column was calibratedby passing cytochrome C (12000Da) carbonic anhydrase(24000Da) bovine serum albumin (66000Da) alcoholdehydrogenase (15000Da) and beta-amylase (200000Da) Acalibration curvewas constructed by plotting logarithmof themolecular weight of the individual marker against the ratio ofthe void (vo) and respective elution volumes (ve) A sampleof B subtilis GGT was passed through the calibrated columnand its vevo ratio was used to calculate the molecular weightfrom the calibration curveThe columnwas then equilibratedwith 100mMCAPSbuffer pH 110 and 150mMNaCl andusedto estimate the molecular weight of B subtilisGGT as before

28 Reversibility Study A sample of the enzyme was incu-bated in 50mM CAPS buffer pH 110 and 150mM NaCl for




ln 119896cat = minus119864119886

119877119879

+ ln119860 (4)



119886119877


ln119896cat119879

= ln 119896119861ℎ

+

Δ119878Dagger

119877

minus

Δ119867Dagger

119877119879

(5)




Δ119866Dagger= Δ119867


Dagger (6)

3 Results


32 119870119898




119898of B subtilis



0

005

01

015

02

025

09

14

19

24

29

7 8 9 10 11

(mic

rom

oles

min

)

pH






0 20 40 60 80 100 120 140

0

50

100

150

200

250

300

Substrate (mM)

(mic

rom

oles

s)

(a)

0 20 40 60 80 100 120 140

0

50

100

150

200

Substrate (mM)

(mic

rom

oles

s)

(b)



pHActivation energy

(E119886)

KJmol




75 239 216 minus1477 656110 279 254 minus1061 571



4 Discussion















References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




ln 119896cat = minus119864119886

119877119879

+ ln119860 (4)



119886119877


ln119896cat119879

= ln 119896119861ℎ

+

Δ119878Dagger

119877

minus

Δ119867Dagger

119877119879

(5)




Δ119866Dagger= Δ119867


Dagger (6)

3 Results


32 119870119898




119898of B subtilis



0

005

01

015

02

025

09

14

19

24

29

7 8 9 10 11

(mic

rom

oles

min

)

pH






0 20 40 60 80 100 120 140

0

50

100

150

200

250

300

Substrate (mM)

(mic

rom

oles

s)

(a)

0 20 40 60 80 100 120 140

0

50

100

150

200

Substrate (mM)

(mic

rom

oles

s)

(b)



pHActivation energy

(E119886)

KJmol




75 239 216 minus1477 656110 279 254 minus1061 571



4 Discussion















References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 20 40 60 80 100 120 140

0

50

100

150

200

250

300

Substrate (mM)

(mic

rom

oles

s)

(a)

0 20 40 60 80 100 120 140

0

50

100

150

200

Substrate (mM)

(mic

rom

oles

s)

(b)



pHActivation energy

(E119886)

KJmol




75 239 216 minus1477 656110 279 254 minus1061 571



4 Discussion















References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

SharathBalakrishnaandAsmitaPrabhunedownloads.hindawi.com/journals/tswj/2014/216270.pdf · respective reaction was determined at , , , and C. Arrhenius plot was used to calculate the