
 peptidomimetics have found wide application as bioavailable , biostable , and potent mimetics of naturally occurring biologically active peptides .
l - arginine has guanidinium group , which is positively charged at neutral ph and is involved in many important physiological and pathophysiological processes .
it has a very ionizable amino acid , and it is found most frequently buried in the protein interior [ 25 ] .
arginine residues are essential in a variety of biological processes , such as the regulation of conformation or redox potentials [ 6 , 7 ] ; viral capsid assembly ; electrostatic steering ; voltage sensing across lipid bilayers [ 1012 ] ; h+ transport [ 6 , 1315 ] and peptide translocation across bilayers [ 16 , 17 ] .
they also play a critical role at protein - protein interfaces [ 5 , 16 ] , in enzymatic active sites [ 3 , 5 , 18 ] , and in variety of transport channels [ 19 , 20 ] .
more recent findings show that arginine - specific methylation of histones may cooperate with other types of posttransitional histone modification to regulate chromatin structure and gene transcription .
proteins that methylate histones on arginine residues can collaborate with other coactivators such as nuclear receptors .
they constitute nature 's toolkit for making and breaking down the molecules required by cells in the course of growth , repair , maintenance , and death .
enzymes are capable of carrying out complex transformations in aqueous solution , at biological temperatures and ph , and in a stereospecific and regiospecific manner , a feat seldom achieved by the best of organic chemists [ 3 , 22 ] .
crystallographic and nmr studies of enzymes have shed light on the relationship between an enzyme 's three - dimensional ( 3d ) structure and the chemical reaction it performs .
there are many complications , however , in assigning the functions of catalytic residues , due to the multistep nature of a chemical reaction .
one residue can play more than one role and can be involved in different steps of the reaction .
arginine ( arg ) is one of the most important residues in catalytic centers of many enzymes .
arg is in the 3rd place of the enzymatic frequency distribution and constitutes 11% of catalytic residues .
arg occurs more frequently than other basic residues ( e.g. , lysine ) because it has three nitrogen containing groups in the side chain , all of which can perform electrostatic interactions .
the side chain of arg can participate therefore in many electrostatic interactions , and it can be positioned more accurately to facilitate catalysis .
the arg side chain has also a favorable geometry to stabilize a pair of oxygen atoms on a phosphate group ( figure 1 ) , a common biological moiety .
arg in a catalytic center can be involved in various kinds of interactions , for example , electrostatic , hydrogen bond formation , transition state stabilization , activation of water , and the activation of substrates .
if the arg residue in the active site of the enzymes is replaced by an arg mimetic , this will cause the loss of enzymatic action , thus disturbing many metabolic pathways .
adenylate kinase ( adk ) was chosen as an example in order to explore how arg analogues will affect enzymatic action .
this enzyme catalyzes the reversible reaction 
 ( 1)2  adp 
 atp+amp 
 and may process metabolic signals associated with atp use [ 2328 ] . in this case , adk has been implicated in the regulation of the metabolically sensitive ion channels and transporters [ 2932 ] . in addition , disruption of the adk gene impedes the export of atp from the mitochondria . what will happen if a gene is working but some mutation of adk appears , such as arginine analogues ( cav , ncav , ncan , nsarg , and sarg ) being incorporated instead of arg in the active site of the enzyme ? in order to study this issue , docking of analogues with arginyl - trna synthetase was our choice for the first step .
this should show if analogues could be recognized by the enzyme responsible for transportation of arginine residues .
aminoacyl - trna constitutes a family of rna - binding proteins , that is , responsible for the correct translation of the genetic code by covalently linking the appropriate amino acid to the 3 end of the correct trna . in most organisms ,
there are 20 distinct aminoacyl - trnas , with each one of them being responsible for aminoacylating its cognate trna with unique amino acid in a two - step catalytic reaction .
this intermediate interacts with appropriate trna molecule , and amino acid is transferred to 3 end of the trna .
the protein sequence for human arginyl - trnasynthetase ( hargs ) was obtained from the uniprot database ( accession number q5t160 ) .
oxy- and sulfoarginine analogues : l - canavanine ( cav ) , l - norcanavanine ( ncav ) , l - norcanaline ( ncan ) , l - norsulfoarginine ( nsarg ) and l - sulfoarginine ( sarg ) , were previously synthesized and biologically tested [ 3639 ] ( figure 2 ) . in order to perform computational studies ,
the following software was used in the present work : compound preparation and homology modeling were done with molecular operating environment ( moe ) ; docking studies were performed by using genetic optimization for ligand docking ( gold 5.1 ) , run on the scientific linux 5.5 operating system ; for generation of figures and the exploration of interactions after docking , the molegro molecular viewer ( mmv ) [ 43 , 44 ] was applied .  
the best hit was proved with the crystallographic structure of arginyl - trna synthetase ( args ) of saccharomyces cerevisiae ( protein data bank -pdb i d : 1f7u ) with 40% identity .
five oxy- and sulfoanalogues of arginine with already known in vivo and in vitro biological effects were selected for docking studies .
it uses a genetic algorithm and considers full ligand conformational flexibility and partial protein flexibility .
the active site of hargs was defined as residues within 10   radius of glu130 , which is responsible for guanidinium group recognition .
one arginine residue seems to be very important at the active site of the enzyme 
arg175 is very close to the active site , and it could play a role in catalytic ability of the enzyme .
docking studies were performed with all mutated enzymes and bis(adenosine)-5-tetraphosphate in order to find out how mutations affect enzymatic action .
the structure of adenylate kinase was obtained from rcsb ( pdb i d : 2c9y ) .
homology modeling of hargs with a single template was performed by moe , and a standard molecular mechanics forcefield - amber99   was used .
phi - psi plot or ramachandran plot for the chosen model is presented on figure 3 .
disallowed regions are less than 1% , according to the ramachandran plot , so it could be used in further studies .
it is known   that atp binds to the loop close to the binding site of arginine .
when there is no atp molecule attached to hargs , arginine binds as shown in figure 4(b ) .
all the h - bonding capability of the substrate is used by the protein for the specific recognition .
the -amino group of arginine forms h - bonds with main chain carbonyls of ser133 and phe134 , while -carboxylate interacts with the amide nitrogen of asn135 and phenyl oxygen of tyr322 .
residues ser133 and asn135 are very important for correct recognition of -amino and -carboxyl groups .
the guanidinium group forms two salt bridges with two carboxylate residues : glu130 and asp326 .
if atp binds , some conformational changes occur , and -carboxyl group of arginine interacts with arg325 , and this facilitates forming the arginyl - amp ( figure 4(c ) ) .
dockings with oxy- and sulfoanalogues of arginine were performed in order to check if they can act as substrates for this enzyme .
the distance between its -carboxyl group and arg326 is too long to interact with it and thus to react successfully with atp ( figure 5(a ) and table 2 ) .
nsarg has a sulfoguanidinium group , and though it is less polar than arginine 's guanidinium group , interaction with glu130 is still present . in the case of this compound
the carboxyl group interacts with other residues from the active site , and the distance between this group and arg325 is too long ( figure 5(b ) ) . in the case of sarg ,
the possibility for interaction between sarg and atp is higher than nsarg as its -carboxyl group is closer to arg325 ( figure 5(b ) ) .
the enzyme can recognize ncav and cav as substrates , but only cav could interact with atp to form cav - amp .
the distance between -carboxyl group and arg325 is too long to have a reaction with atp ( figure 5(a ) ) . from this series of arginine analogues
, we could mark three of them that could play the role of substrates for hargs .
these compounds could be incorporated in different peptides and protein molecules , and in the present work , we are analyzing the effects of this interaction on adenylate kinase . with the help of moe , we made mutations at two points subsequently in the active site of adk - arg138 and arg175 , with cav , ncav , and sarg .
after energy minimizations of 6 new structures , docking with bis(adenosine)-5-tetraphosphate was performed with gold .
bis(adenosine)-5-tetraphosphate could mimic the interactions in the binding sites of adk as it occupies both sites of the enzyme . for the recognition of purine moiety of the substrate , very important residue
it interacts with all phosphate oxygen atoms of adp molecule by forming hydrogen bonds with backbone amide groups ( figure 6 ) .
all three analogues of arginine have the guanidinium group as a structural element . in all three cases ,
it is connected via more electronegative atoms ( oxygen and sulfur ) than the carbon atom ; thus , it is less polar than guanidinium group of arg . in all three mutations of arg138 with its analogues ,
the typical site for adp recognition still remains , and the purine moiety binds respectively , to oxy- , and sulfoguanidinium group . in the cases of cav138 and sarg138 mutations ,
total energies of the enzyme - substrate complexes decrease , while in the case of cav138 mutation , total energy increases .
fitness function value is almost the same when arg138 is replaced by cav , due to their structural similarity , and conformational changes in that case are very small ( figures 7(a ) and 7(b ) ) .
when arg138 is replaced by ncav and sarg , conformational changes in the binding site of the enzyme are bigger , and fitness function values are higher which is connected to the binding affinity of the substrate .
the higher the fitness function value , the higher the affinity of the substrate to the enzyme and it will bind more strongly . this will decrease the rate of the enzymatic reaction and most probably will stop it in the case of ncan138 and sarg138 .
there are some conformational changes and the values of fitness function indicate that , but newly formed enzyme - substrate complexes have total energies not very different from that of wild type enzyme - substrate complex .
these mutations do not affect arg138 and the binding site for purine moiety remains unchanged ( figure 7(c ) ) .
exploring the sequence gagkg in the case of arg138 mutation , the biggest conformational changes occurred when arg138 is replaced by ncav ( figure 8(a ) ) . in all mutations in position 175 ,
purine moiety could be recognized by the mutant enzyme , but because of the changes in the site responsible for interaction between two adp molecules , reaction could not be performed .
from previously synthesized and biologically tested arginine analogues , three of them cav , ncav , and sarg could act as its antimetabolites .
they could be recognized by arginylate - trna synthetase as substrates and could be included in numerous biologically important peptides and proteins .
once they become an element of proteins ' sequence , for example , enzymes , they would cause crucial changes in their structure leading to loss of enzymatic action .
as long as these compounds exhibit a prolonged biological effect , this most probably is due to their incorporation into important metabolic enzymes .
 arginine residues are very important for the structure of proteins and their action . 
 arginine is essential for many natural processes because it has unique ionizable group under physiological conditions . 
 numerous mimetics of arginine were synthesized and their biological effects were evaluated , but the mechanisms of actions are still unknown . 
 the aim of this study is to see if oxy- and sulfoanalogues of arginine can be recognized by human arginyl - trna synthetase ( hargs)an enzyme responsible for coupling of l - arginine with its cognate trna in a two - step catalytic reaction . 
 we make 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 . 
 three analogues of arginine , l - canavanine ( cav ) , l - norcanavanine ( ncav ) , and l - sulfoarginine ( sarg ) , can be recognized as substrates of hargs when incorporated in different peptide and protein sequences instead of l - arginine . 
 mutation in the enzyme active center by arginine mimetics leads to conformational changes , which produce a decrease the rate of the enzyme catalyzed reaction and even a loss of enzymatic action . 
 all these observations could explain the long - lasting nature of the effects of the arginine analogues . 