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Hindawi Publishing CorporationJournal of Amino AcidsVolume 2013 Article ID 407616 7 pageshttpdxdoiorg1011552013407616

Research ArticleLong-Lasting Effects of Oxy- and Sulfoanalogues ofl-Arginine on Enzyme Actions

Tatyana A Dzimbova1 Peter B Milanov23 and Tamara I Pajpanova1

1 Institute of Molecular Biology Bulgarian Academy of Sciences 1113 Sofia Bulgaria2 Faculty of Mathematics and Natural Sciences South-West University ldquoNeofit Rilskirdquo 2700 Blagoevgrad Bulgaria3 Institute of Mathematics and Informatics Bulgarian Academy of Sciences 1113 Sofia Bulgaria

Correspondence should be addressed to Tatyana A Dzimbova tania dzimbovaabvbg

Received 5 June 2013 Revised 20 August 2013 Accepted 15 September 2013

Academic Editor Hieronim Jakubowski

Copyright copy 2013 Tatyana A Dzimbova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Arginine residues are very important for the structure of proteins and their action Arginine is essential for many natural processesbecause it has unique ionizable group under physiological conditions Numerous mimetics of arginine were synthesized and theirbiological effects were evaluated but the mechanisms of actions are still unknown The aim of this study is to see if oxy- andsulfoanalogues of arginine can be recognized by human arginyl-tRNA synthetase (HArgS)mdashan enzyme responsible for coupling ofl-arginine with its cognate tRNA in a two-step catalytic reactionWemake use of modeling and docking studies of adenylate kinase(ADK) to reveal the effects produced by the incorporation of the arginine mimetics on the structure of ADK and its action Threeanalogues of arginine l-canavanine (Cav) l-norcanavanine (NCav) and l-sulfoarginine (sArg) can be recognized as substrates ofHArgS when incorporated in different peptide and protein sequences instead of l-arginine Mutation in the enzyme active centerby arginine mimetics leads to conformational changes which produce a decrease the rate of the enzyme catalyzed reaction andeven a loss of enzymatic action All these observations could explain the long-lasting nature of the effects of the arginine analogues

1 Introduction

Peptidomimetics have foundwide application as bioavailablebiostable and potent mimetics of naturally occurring bio-logically active peptides l-Arginine has guanidinium groupwhich is positively charged at neutral pH and is involved inmany important physiological and pathophysiological pro-cesses [1] It has a very ionizable amino acid and it is foundmost frequently buried in the protein interior [2ndash5] Arginineresidues are essential in a variety of biological processes suchas the regulation of conformation or redox potentials [6 7]viral capsid assembly [8] electrostatic steering [9] voltagesensing across lipid bilayers [10ndash12] H+ transport [6 13ndash15] and peptide translocation across bilayers [16 17] Theyalso play a critical role at protein-protein interfaces [5 16]in enzymatic active sites [3 5 18] and in variety of transportchannels [19 20] More recent findings show that arginine-specific methylation of histones may cooperate with othertypes of posttransitional histone modification to regulate

chromatin structure and gene transcription [21] Proteins thatmethylate histones on arginine residues can collaborate withother coactivators such as nuclear receptors

Enzymes are probably the most studied biologicalmolecules They constitute naturersquos toolkit for making andbreaking down the molecules required by cells in thecourse of growth repair maintenance and death Virtuallyevery biological process requires an enzyme at some pointEnzymes are capable of carrying out complex transforma-tions in aqueous solution at biological temperatures and pHand in a stereospecific and regiospecificmanner a feat seldomachieved by the best of organic chemists [3 22] Crystal-lographic and NMR studies of enzymes have shed light onthe relationship between an enzymersquos three-dimensional (3D)structure and the chemical reaction it performs There aremany complications however in assigning the functions ofcatalytic residues due to the multistep nature of a chemicalreaction One residue can play more than one role and can beinvolved in different steps of the reaction

2 Journal of Amino Acids

N N

NRH

H

HH

H

C

O

O

O O

O

120575+

120575+

120575+

Figure 1 Schematic presentation of the hydrogen bond formationof the guanidinium group with 5 different oxygen atoms

Arginine (Arg) is one of the most important residuesin catalytic centers of many enzymes Arg is in the 3rdplace of the enzymatic frequency distribution and constitutes11 of catalytic residues [3] Arg occurs more frequentlythan other basic residues (eg lysine) because it has threenitrogen containing groups in the side chain all of which canperform electrostatic interactions The side chain of Arg canparticipate therefore inmany electrostatic interactions and itcan be positioned more accurately to facilitate catalysis TheArg side chain has also a favorable geometry to stabilize a pairof oxygen atoms on a phosphate group (Figure 1) a commonbiological moiety [3] Arg in a catalytic center can be involvedin various kinds of interactions for example electrostatichydrogen bond formation transition state stabilization acti-vation of water and the activation of substrates

If the Arg residue in the active site of the enzymes isreplaced by an Arg mimetic this will cause the loss of enzy-matic action thus disturbing many metabolic pathways Thisprobably could be the reason for cell disorders Adenylatekinase (ADK) was chosen as an example in order to explorehow Arg analogues will affect enzymatic actionThis enzymecatalyzes the reversible reaction

2ADP 999447999472 ATP + AMP (1)

and may process metabolic signals associated with ATPuse [23ndash28] In this case ADK has been implicated in theregulation of the metabolically sensitive ion channels andtransporters [29ndash32] In addition disruption of the ADKgene impedes the export of ATP from the mitochondria [33]What will happen if a gene is working but some mutation ofADK appears such as arginine analogues (Cav NCav NCanNsArg and sArg) being incorporated instead of Arg in theactive site of the enzyme

In order to study this issue docking of analogues witharginyl-tRNA synthetase was our choice for the first stepThis should show if analogues could be recognized by theenzyme responsible for transportation of arginine residues[34] Aminoacyl-tRNA constitutes a family of RNA-bindingproteins that is responsible for the correct translation ofthe genetic code by covalently linking the appropriate aminoacid to the 31015840 end of the correct tRNA In most organismsthere are 20 distinct aminoacyl-tRNAs with each one ofthem being responsible for aminoacylating its cognate tRNAwith unique amino acid in a two-step catalytic reaction

O ONH

NH

NH2

OH

O

NH

NH

NH2

O OH

NH2

OS

NH

NH

OHO O

SNH

NH

NH2

O O

NH2

O

OH

O

O

OH

Cav NCav NCan

sArg NsArg

H2N

H2N

H2N H2N

H2N

Figure 2 Structures of the investigated arginine analogues

Psi

General180

150

120

90

60

30

0

minus30

minus60

minus90

minus120

minus150

minus180minus150 minus90 minus30 30 90 150

Phi

Figure 3 Ramachandran plot of HArgS obtained from MOEmost favorable regions are minus808 allowed regions are minus183 anddisallowed regions are minus09 outliers were presented with +

[35] The mechanism of their action is well known Thesynthetase first binds ATP and corresponding amino acidto form acetylamino-AMP This intermediate interacts withappropriate tRNAmolecule and amino acid is transferred to31015840 end of the tRNA

2 Methods

21 Enzymes and Arginine Analogues The protein sequencefor human arginyl-tRNAsynthetase (HArgS) was obtainedfrom the UniProt database (accession number Q5T160)[34] Oxy- and sulfoarginine analogues l-canavanine (Cav)l-norcanavanine (NCav) l-norcanaline (NCan) l-norsulfo-arginine (NsArg) and l-sulfoarginine (sArg) were previouslysynthesized and biologically tested [36ndash39] (Figure 2)

22 Computational Tools In order to perform computationalstudies the following software was used in the present workcompound preparation and homology modeling were done

Journal of Amino Acids 3

(a) (b) (c)

Figure 4 Binding of ATP to HArgS (a) l-arginine to HArgS without ATP (b) and l-arginine to HArgS with ATPc

(a) (b) (c)

Figure 5 Arginine analogues superposed in the active site arginine dark green-Cav light green-NCan dark blue-NCav light blue-NsArgand purple-sArg

with Molecular Operating Environment (MOE) [40] dock-ing studies were performed by using Genetic Optimizationfor Ligand Docking (GOLD 51) [41] run on the ScientificLINUX 55 operating system [42] for generation of figuresand the exploration of interactions after docking theMolegroMolecular Viewer (MMV) [43 44] was applied

23 Homology Modeling It was based on a single templateand was performed with MOE The best hit was proved withthe crystallographic structure of arginyl-tRNA synthetase(ArgS) of Saccharomyces cerevisiae (Protein Data Bank [45]-PDB Id 1f7u) with 40 identity

24 Docking of Arginine Analogues Five oxy- and sulfoana-logues of argininewith already known in vivo and in vitro bio-logical effects were selected for docking studies 3D structuresof the compounds were modeled with MOE and protonatedat physiological pH Docking was carried out with GOLD 51software It uses a genetic algorithm and considers full ligandconformational flexibility and partial protein flexibility Theactive site of HArgS was defined as residues within 10 A

radius of Glu130 which is responsible for guanidinium grouprecognition

25 Mutations of Adenylate Kinase (ADK) One arginineresidue seems to be very important at the active site ofthe enzymemdashArg138 Arg175 is very close to the active siteand it could play a role in catalytic ability of the enzymeMutations were made in MOE by redrawing the residuesand minimizing structures obtained Docking studies wereperformed with all mutated enzymes and bis(adenosine)-51015840-tetraphosphate in order to find out how mutations affectenzymatic action The structure of adenylate kinase wasobtained from RCSB (PDB Id 2c9y)

3 Results and Discussion

31 Modeling and Docking of HArgS Homology modelingof HArgS with a single template was performed by MOEand a standard molecular mechanics forcefield-amber99[46] was used Ten models were generated and the modelwas optimized by energy minimization Phi-psi plot or


Table 1 Fitness function values interactions with the enzyme total energies and energies of binding with the 120572-carboxyl group of thecompounds investigated

Compound Fitness function Interactions with the enzyme

Arg 464Two interactions between Glu130-Gu group and Asp326-Gu groupand 2 interactions between Arg325-120572-carboxyl groupGlu269-120572-carboxyl group Gly172-120572-amino group

NCan 3298 Glu130-oxyamino group Asp326-120572-amino group andHis353-120572-carboxyl group

NCav 4409 Two interactions between Glu130-oxyGu group Tyr322Asn135-120572-carboxyl group Tyr170 and Ser133-120572-amino group

Cav 368 Two interactions between Glu130-oxyGu group Tyr322 Asp135Gly172-120572-carboxyl group and Leu171-120572-amino group

NsArg 4404Glu130 Asp326-sulfoGu group Asp326 Tyr344-sulfoGu groupTyr322-SO2 group Ser133-120572-amino group Glu269 andAsn135-120572-carboxyl group

sArg 4678 Two interactions between Glu130-sulfoGu group Tyr322-SO2 groupAsp135 Gly172-120572-carboxyl group and Ser133-120572-amino group

Figure 6 Interactions in the active site of ADKwith bis(adenosine)-51015840-tetraphosphate

Ramachandran plot for the chosen model is presented onFigure 3

Disallowed regions are less than 1 according to theRamachandran plot so it could be used in further studiesThe selected model was also checked by docking

It is known [47] that ATP binds to the loop close to thebinding site of arginineThis helps the forming of the arginyl-AMP It interacts electrostatically with Arg325 (Figure 4(a))When there is no ATP molecule attached to HArgS argininebinds as shown in Figure 4(b) All the H-bonding capabilityof the substrate is used by the protein for the specificrecognition The 120572-amino group of arginine forms H-bondswith main chain carbonyls of Ser133 and Phe134 while 120572-carboxylate interacts with the amide nitrogen of Asn135 andphenyl oxygen of Tyr322 Residues Ser133 andAsn135 are veryimportant for correct recognition of 120572-amino and 120572-carboxylgroups The guanidinium group forms two salt bridges withtwo carboxylate residues Glu130 and Asp326 If ATP bindssome conformational changes occur and 120572-carboxyl groupof arginine interacts with Arg325 and this facilitates formingthe arginyl-AMP (Figure 4(c))

Table 2 Total energies of the complexes and fitness function valuesof bis(adenosine)-51015840-tetraphosphate with ADK and mutated ADK

Mutation Total energy ofthe complex

Fitness functionvalue

Without any mutationArg138 and Arg175 minus135483 6929

Cav138 Arg175 minus112329 6808NCav138 Arg175 minus147651 9030sArg138 Arg175 minus102358 9774Arg138 Cav175 minus128855 6808Arg138 NCav175 minus127472 9376Arg138 sArg175 minus131506 9624

Dockings with oxy- and sulfoanalogues of arginine wereperformed in order to check if they can act as substrates forthis enzyme

The interactions between the compounds and enzymeare listed in Table 1 Glu130 is an important site for substraterecognition in the enzyme It binds to the guanidinium groupof arginine In themolecule ofNCan there is no guanidiniumgroup and such interaction does not appear Fitness functionvalue for NCan is the lowest in this series of compoundsThedistance between its 120572-carboxyl group and Arg326 is too longto interact with it and thus to react successfully with ATP(Figure 5(a) and Table 2)

NsArg has a sulfoguanidinium group and though it isless polar than argininersquos guanidiniumgroup interactionwithGlu130 is still present In the case of this compound we haveadditional interaction of the sulfogroup with Tyr322 Thecarboxyl group interacts with other residues from the activesite and the distance between this group and Arg325 is toolong (Figure 5(b))

In the case of sArg the possibility for interaction betweensArg and ATP is higher than NsArg as its 120572-carboxyl group iscloser to Arg325 (Figure 5(b))


B

(a) (b) (c)

Figure 7 Superposed active sites of ADK and Arg138 mutated ADK with bis(adenosine)-51015840-tetraphosphate (a) focus on Arg138 residue inArg138 mutated enzymes (b) superposed active sites of ADK and Arg175 mutated ADK with bis(adenosine)-51015840-tetraphosphate (c)

(a) (b)

Figure 8 Superposition of the sequence GAGKG (25ndash29) (a) ADK and ADK mutated with Cav138 (blue) NCav138 (green) and sArg138(purple) (b) ADK and ADK mutated with Cav175 (blue) NCav175 (green) and sArg175 (purple)

The enzyme can recognize NCav and Cav as substratesbut only Cav could interact with ATP to form Cav-AMPThedistance between 120572-carboxyl group and Arg325 is too long tohave a reaction with ATP (Figure 5(a))

From this series of arginine analogues we could markthree of them that could play the role of substrates for HArgSThese compounds could be incorporated in different peptidesand protein molecules and in the present work we areanalyzing the effects of this interaction on adenylate kinase

32 Docking of Mutant Adenylate Kinases (ADK) With thehelp of MOE we made mutations at two points subsequentlyin the active site of ADK-Arg138 and Arg175 with CavNCav and sArg After energy minimizations of 6 new struc-tures docking with bis(adenosine)-51015840-tetraphosphate wasperformed with GOLD Bis(adenosine)-51015840-tetraphosphatecould mimic the interactions in the binding sites of ADK asit occupies both sites of the enzyme

For the recognition of purinemoiety of the substrate veryimportant residue is needed Arg138 which interacts by a p-120587interaction

Second important point in enzyme sequence is GAGKG(residues 25ndash29) It is conserved and fully closed over sub-strate molecule It interacts with all phosphate oxygen atomsof ADPmolecule by forming hydrogen bonds with backboneamide groups (Figure 6)

All three analogues of arginine have the guanidiniumgroup as a structural element In all three cases it is connectedvia more electronegative atoms (oxygen and sulfur) than thecarbon atom thus it is less polar than guanidinium group ofArg In all three mutations of Arg138 with its analogues thetypical site for ADP recognition still remains and the purinemoiety binds respectively to oxy- and sulfoguanidiniumgroup In the cases of Cav138 and sArg138 mutations totalenergies of the enzyme-substrate complexes decrease whilein the case of Cav138mutation total energy increases Fitnessfunction value is almost the same when Arg138 is replacedby Cav due to their structural similarity and conformationalchanges in that case are very small (Figures 7(a) and 7(b))When Arg138 is replaced by NCav and sArg conformationalchanges in the binding site of the enzyme are bigger andfitness function values are higher which is connected tothe binding affinity of the substrate The higher the fitnessfunction value the higher the affinity of the substrate to theenzyme and it will bind more strongly This will decrease therate of the enzymatic reaction and most probably will stop itin the case of NCan138 and sArg138

Mutations in position Arg175 do not affect the enzymemuchThere are some conformational changes and the valuesof fitness function indicate that but newly formed enzyme-substrate complexes have total energies not very differentfrom that of wild type enzyme-substrate complex These


mutations do not affect Arg138 and the binding site for purinemoiety remains unchanged (Figure 7(c))

Exploring the sequence GAGKG in the case of Arg138mutation the biggest conformational changes occurredwhenArg138 is replaced by NCav (Figure 8(a))

In all mutations in position 175 conformational changesare significant (Figure 8(b)) Purine moiety could be recog-nized by themutant enzyme but because of the changes in thesite responsible for interaction between two ADP moleculesreaction could not be performedThese mutations could leadto inactive enzyme

4 Conclusion

From previously synthesized and biologically tested arginineanalogues three of them Cav NCav and sArg could act asits antimetabolites They could be recognized by arginylate-tRNA synthetase as substrates and could be included innumerous biologically important peptides and proteinsOnce they become an element of proteinsrsquo sequence forexample enzymes they would cause crucial changes in theirstructure leading to loss of enzymatic action As long asthese compounds exhibit a prolonged biological effect thismost probably is due to their incorporation into importantmetabolic enzymes

Acknowledgments

This work was supported by NFSR of Bulgaria ProjectDVU 01197 and COST Action CM0801 Project DO 02-13531072009 Special thanks are due to Professor EdwardWRandall Queen Mary University of London for the proof-reading and editing of the paper

References

[1] L P Masic ldquoArginine mimetic structures in biologically activeantagonists and inhibitorsrdquo Current Medicinal Chemistry vol13 no 30 pp 3627ndash3648 2006

[2] M J Harms J L SchlessmanG R Sue and E BertrandGarcıa-Moreno ldquoArginine residues at internal positions in a protein arealways chargedrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 108 no 47 pp 18954ndash189592011

[3] G J Bartlett C T Porter N Borkakoti and J M ThorntonldquoAnalysis of catalytic residues in enzyme active sitesrdquo Journal ofMolecular Biology vol 324 no 1 pp 105ndash121 2002

[4] J Kim JMao andMRGunner ldquoAre acidic and basic groups inburied proteins predicted to be ionizedrdquo Journal of MolecularBiology vol 348 no 5 pp 1283ndash1298 2005

[5] A A Bogan and K S Thorn ldquoAnatomy of hot spots in proteininterfacesrdquo Journal of Molecular Biology vol 280 no 1 pp 1ndash91998

[6] R L Cutler AM Davies S Creighton et al ldquoRole of Arginine-38 in regulation of the cytochrome c oxidation-reductionequilibriumrdquo Biochemistry vol 28 no 8 pp 3188ndash3197 1989

[7] P J Winn S K Ludemann R Gauges V Lounnas and RC Wade ldquoComparison of the dynamics of substrate accesschannels in three cytochrome p450s reveals different opening

mechanisms and a novel functional role for a buried argininerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 99 no 8 pp 5361ndash5366 2002

[8] J L Hansen A M Long and S C Schultz ldquoStructure of theRNA-dependent RNA polymerase of poliovirusrdquo Structure vol5 no 8 pp 1109ndash1122 1997

[9] R C Wade R R Gabdoulline S K Ludemann and VLounnas ldquoElectrostatic steering and ionic tethering in enzyme-ligand binding insights from simulationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 95 no 11 pp 5942ndash5949 1998

[10] Y Jiang V Ruta J Chen A Lee and R MacKinnon ldquoTheprinciple of gating chargemovement in a voltage-dependent K+channelrdquo Nature vol 423 no 6935 pp 42ndash48 2003

[11] S B Long E B Campbell and R MacKinnon ldquoVoltagesensor of Kv12 structural basis of electromechanical couplingrdquoScience vol 309 no 5736 pp 903ndash908 2005

[12] X Tao A Lee W Limapichat D A Dougherty and RMacKinnon ldquoA gating charge transfer center in voltage sensorsrdquoScience vol 328 no 5974 pp 67ndash73 2010

[13] N P Le H Omote Y Wada M K Al-Shawi R K Nakamotoand M Futai ldquoEscherichia coli ATP synthase 120572 subunit Arg-376 the catalytic site arginine does not participate in thehydrolysissynthesis reaction but is required for promotion tothe steady staterdquo Biochemistry vol 39 no 10 pp 2778ndash27832000

[14] R Rammelsberg G HuhnM Lubben andK Gerwert ldquoBacte-riorhodopsinrsquos intramolecular proton-release pathway consistsof a hydrogen-bonded networkrdquoBiochemistry vol 37 no 14 pp5001ndash5009 1998

[15] H Luecke H-T Richter and J K Lanyi ldquoProton transferpathways in bacteriorhodopsin at 23 angstrom resolutionrdquoScience vol 280 no 5371 pp 1934ndash1937 1998

[16] S Futaki T Suzuki W Ohashi et al ldquoArginine-rich peptidesAn abundant source of membrane-permeable peptides havingpotential as carriers for intracellular protein deliveryrdquo Journal ofBiological Chemistry vol 276 no 8 pp 5836ndash5840 2001

[17] H D Herce and A E Garcia ldquoMolecular dynamics simulationssuggest a mechanism for translocation of the HIV-1 TATpeptide across lipid membranesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no52 pp 20805ndash20810 2007

[18] P B Crowley andAGolovin ldquoCation-120587 interactions in protein-protein interfacesrdquo Proteins vol 59 no 2 pp 231ndash239 2005

[19] D F Savage J D OrsquoConnell III L JWMiercke J Finer-Mooreand R M Stroud ldquoStructural context shapes the aquaporinselectivity filterrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 107 no 40 pp 17164ndash171692010

[20] O Boudker R M Ryan D Yernool K Shimamoto and EGouaux ldquoCoupling substrate and ion binding to extracellulargate of a sodium-dependent aspartate transporterrdquo Nature vol445 no 7126 pp 387ndash393 2007

[21] M R Stallcup ldquoRole of protein methylation in chromatinremodeling and transcriptional regulationrdquo Oncogene vol 20no 24 pp 3014ndash3020 2001

[22] C Walsh ldquoEnabling the chemistry of liferdquo Nature vol 409 no6817 pp 226ndash231 2001

[23] P P Dzeja K T Vitkevicius M M Redfield J C Burnettand A Terzic ldquoAdenylate kinase-catalyzed phosphotransferin the myocardium increased contribution in heart failurerdquoCirculation Research vol 84 no 10 pp 1137ndash1143 1999


[24] S P Bessman and C L Carpenter ldquoThe creatine-creatinephosphate energy shuttlerdquo Annual Review of Biochemistry vol54 pp 831ndash862 1985

[25] P P Dzeja R J Zeleznikar and N D Goldberg ldquoAdenylatekinase kinetic behavior in intact cells indicates it is integralto multiple cellular processesrdquoMolecular and Cellular Biochem-istry vol 184 no 1-2 pp 169ndash182 1998

[26] P Dzeja A Kalvenas A Toleikis and A Praskevicius ldquoTheeffect of adenylate kinase activity on the rate and efficiency ofenergy transport from mitochondria to hexokinaserdquo Biochem-istry International vol 10 no 2 pp 259ndash265 1985

[27] S Kubo and L H Noda ldquoAdenylate kinase of porcine heartrdquoEuropean Journal of Biochemistry vol 48 no 2 pp 325ndash3311974

[28] F D Laterveer K Nicolay and F N Gellerich ldquoExperimen-tal evidence for dynamic compartmentation of ADP at themitochondrial periphery coupling of mitochondrial adenylatekinase and mitochondrial hexokinase with oxidative phospho-rylation under conditions mimicking the intracellular colloidosmotic pressurerdquoMolecular and Cellular Biochemistry vol 174no 1-2 pp 43ndash51 1997

[29] P P Dzeja and A Terzic ldquoPhosphotransfer reactions in theregulation of ATP-sensitive K+ channelsrdquo FASEB Journal vol12 no 7 pp 523ndash529 1998

[30] J R Elvir-Mairena A Jovanovic L A Gomez A E Alekseevand A Terzic ldquoReversal of the ATP-liganded state of ATP-sensitive K+ channels by adenylate kinase activityrdquo Journal ofBiological Chemistry vol 271 no 50 pp 31903ndash31908 1996

[31] J A Gutierrez and LN Csonka ldquoIsolation and characterizationof adenylate kinase (adk)mutations in Salmonella typhimuriumwhich block the ability of glycine betaine to function as anosmoprotectantrdquo Journal of Bacteriology vol 177 no 2 pp 390ndash400 1995

[32] L Karl OlsonW Schroeder R Paul Robertson NDGoldbergand T F Walseth ldquoSuppression of adenylate kinase catalyzedphosphotransfer precedes and is associated with glucose-induced insulin secretion in intact HIT-T15 cellsrdquo Journal ofBiological Chemistry vol 271 no 28 pp 16544ndash16552 1996

[33] W Bandlow G Strobel C Zoglowek U Oechsner and VMagdolen ldquoYeast adenylate kinase is active simultaneouslyin mitochondria and cytoplasm and is required for non-fermentative growthrdquo European Journal of Biochemistry vol178 no 2 pp 451ndash457 1988

[34] httpwwwuniprotorg[35] BDelagoutteDMoras and J Cavarelli ldquotRNAaminoacylation

by arginyl-tRNA synthetase induced conformations duringsubstrates bindingrdquo EMBO Journal vol 19 no 21 pp 5599ndash5610 2000

[36] T Dzimbova I Iliev K Georgiev R Detcheva A Balachevaand T Pajpanova ldquoIn vitro assessment of the cytotoxic effectsof hydrazide derivatives of unnatural amino acidsrdquo in PeptidesBuilding Bridges Proceedings of the 22nd American PeptideSymposium M Lebl Ed pp 392ndash393 2011

[37] T Dzimbova I Iliev R Detcheva A Balacheva and TPajpanova ldquoIn vitro assessment of the cytotoxic effects ofhydrazide derivatives of sulfoarginines in 3T3 andHepG2 cellsrdquoinProceedings of the Collection Symposium Series vol 13 pp 34ndash36 2011

[38] T Dzimbova E Miladinova S Mohr et al ldquoSulfo- andoxy-analogues of arginine synthesis analysis and preliminarybiological screeningrdquo Croatica Chemica Acta vol 84 no 3 pp447ndash453 2011

[39] T Dzimbova I Iliev K Georgiev R Detcheva A Balachevaand T Pajpanova ldquoIn vitro assessment of the cytotoxic effects ofsulfo-arginine analogues and their hydrazide derivatives in 3T3and HepG2 cellsrdquo Biotechnology amp Biotechnological Equipmentvol 26 pp 180ndash184 2012

[40] Molecular Operating Environment (MOE) 2012 10 ChemicalComputing Group Inc 1010 Sherbooke St West Suite 910Montreal QC Canada H3A 2R7 2012

[41] G Jones P Willett R C Glen A R Leach and R TaylorldquoDevelopment and validation of a genetic algorithm for flexibledockingrdquo Journal of Molecular Biology vol 267 no 3 pp 727ndash748 1997

[42] httpwwwscientificlinuxorg[43] httpmolegrocomindexphp[44] R Thomsen and M H Christensen ldquoMolDock a new tech-

nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[45] httpwwwrcsborg[46] W D Cornell P Cieplak C I Bayly et al ldquoA second generation

force field for the simulation of proteins nucleic acids andorganic moleculesrdquo Journal of the American Chemical Societyvol 117 no 19 pp 5179ndash5197 1995

[47] J Cavarelli B Delagoutte G Eriani J Gangloff and D MorasldquoL-arginine recognition by yeast arginyl-tRNA synthetaserdquoEMBO Journal vol 17 no 18 pp 5438ndash5448 1998

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Enzyme Research



Microbiology


N N

NRH

H

HH

H

C

O

O

O O

O

120575+

120575+

120575+

Figure 1 Schematic presentation of the hydrogen bond formationof the guanidinium group with 5 different oxygen atoms

Arginine (Arg) is one of the most important residuesin catalytic centers of many enzymes Arg is in the 3rdplace of the enzymatic frequency distribution and constitutes11 of catalytic residues [3] Arg occurs more frequentlythan other basic residues (eg lysine) because it has threenitrogen containing groups in the side chain all of which canperform electrostatic interactions The side chain of Arg canparticipate therefore inmany electrostatic interactions and itcan be positioned more accurately to facilitate catalysis TheArg side chain has also a favorable geometry to stabilize a pairof oxygen atoms on a phosphate group (Figure 1) a commonbiological moiety [3] Arg in a catalytic center can be involvedin various kinds of interactions for example electrostatichydrogen bond formation transition state stabilization acti-vation of water and the activation of substrates

If the Arg residue in the active site of the enzymes isreplaced by an Arg mimetic this will cause the loss of enzy-matic action thus disturbing many metabolic pathways Thisprobably could be the reason for cell disorders Adenylatekinase (ADK) was chosen as an example in order to explorehow Arg analogues will affect enzymatic actionThis enzymecatalyzes the reversible reaction

2ADP 999447999472 ATP + AMP (1)

and may process metabolic signals associated with ATPuse [23ndash28] In this case ADK has been implicated in theregulation of the metabolically sensitive ion channels andtransporters [29ndash32] In addition disruption of the ADKgene impedes the export of ATP from the mitochondria [33]What will happen if a gene is working but some mutation ofADK appears such as arginine analogues (Cav NCav NCanNsArg and sArg) being incorporated instead of Arg in theactive site of the enzyme

In order to study this issue docking of analogues witharginyl-tRNA synthetase was our choice for the first stepThis should show if analogues could be recognized by theenzyme responsible for transportation of arginine residues[34] Aminoacyl-tRNA constitutes a family of RNA-bindingproteins that is responsible for the correct translation ofthe genetic code by covalently linking the appropriate aminoacid to the 31015840 end of the correct tRNA In most organismsthere are 20 distinct aminoacyl-tRNAs with each one ofthem being responsible for aminoacylating its cognate tRNAwith unique amino acid in a two-step catalytic reaction

O ONH

NH

NH2

OH

O

NH

NH

NH2

O OH

NH2

OS

NH

NH

OHO O

SNH

NH

NH2

O O

NH2

O

OH

O

O

OH

Cav NCav NCan

sArg NsArg

H2N

H2N

H2N H2N

H2N

Figure 2 Structures of the investigated arginine analogues

Psi

General180

150

120

90

60

30

0

minus30

minus60

minus90

minus120

minus150

minus180minus150 minus90 minus30 30 90 150

Phi

Figure 3 Ramachandran plot of HArgS obtained from MOEmost favorable regions are minus808 allowed regions are minus183 anddisallowed regions are minus09 outliers were presented with +

[35] The mechanism of their action is well known Thesynthetase first binds ATP and corresponding amino acidto form acetylamino-AMP This intermediate interacts withappropriate tRNAmolecule and amino acid is transferred to31015840 end of the tRNA

2 Methods

21 Enzymes and Arginine Analogues The protein sequencefor human arginyl-tRNAsynthetase (HArgS) was obtainedfrom the UniProt database (accession number Q5T160)[34] Oxy- and sulfoarginine analogues l-canavanine (Cav)l-norcanavanine (NCav) l-norcanaline (NCan) l-norsulfo-arginine (NsArg) and l-sulfoarginine (sArg) were previouslysynthesized and biologically tested [36ndash39] (Figure 2)

22 Computational Tools In order to perform computationalstudies the following software was used in the present workcompound preparation and homology modeling were done


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4 Conclusion


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Volume 2014




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4 Conclusion


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4 Conclusion


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Advances in

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Volume 2014




Enzyme Research



Microbiology





4 Conclusion


Acknowledgments


References
























































Volume 2014

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Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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