Studies on sensitivity to racemization of activated residues in couplings of N-benzyloxycarbonyldipeptides.

A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa = 15 different residues and R = H, NH2, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5 degrees. The accompanying racemization was determined by analysis of the epimeric products by normal phase high-performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5 degrees, and N,N'-dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosp hate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.     

Peptide synthesis in organic media with the use of subtilisin 72 immobilized on a poly(vinyl alcohol) cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Peptide Synthesis in Organic Media with the Use of Subtilisin 72 Immobilized on a Poly(Vinyl Alcohol) Cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate.     

Stereoselective acylation of a racemic amine with C(alpha)-methyl phenylglycine-based dipeptide 5(4H)-oxazolones

Reactions of a racemic amine with chiral, N(alpha)-acetylated, C(alpha)-methyl l-phenylglycine-based dipeptide 5(4H)-oxazolones proceed diastereoselectively to give predominantly dipeptide alkylamides comprising d-alpha-phenylethylamine. Diastereoselectivity is remarkably sensitive to solvent polarity and reaction temperature but not significantly to the nature of the C(alpha)-tetrasubstituted alpha-amino acid at position 1 of the dipeptide. The beta-turn 3D structures of the aminolysis products were established in CDCl(3) solution by FT-IR absorption and in one case in the crystal state by X-ray diffraction as well.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

Conformational investigation of alpha,beta-dehydropeptides. Part. IV. Beta-turn in saturated and alpha,beta-unsaturated peptides Ac-Pro-Xaa-NHCH3: NMR and IR studies.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.     

Solvolysis and aminolysis on peptidyl-Kaiser oxime resin assisted by Ca2+ and Eu3+: a mild procedure to prepare alpha-methyl and -ethyl esters of protected peptides.

Ca2+ and Eu3+ were able to assist solvolysis on peptidyl-Kaiser oxime resins generating alpha-methyl and -ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca2+ or Eu3+ was needed to enhance this reaction efficiency. Ca2+ also assisted aminolysis on peptidyl-Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of the C-terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the beta-benzyl or cyclohexyl esters, was detected in the reaction media. The alpha-methyl and -ethyl esters of Ac-Ala-Gly-X [where, X = Gly, Ala, Phe or Lys (2-Cl-Z)] and of Ac-Ile-Ser (Bzl)-Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac-Ile-Ser(Bzl)-Asp(OcHex)Arg(HCl)-OMe and Ac-Ile-Ser(Bzl)-Asp(OcHex)Arg (HCl)-OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin-cleavage yields were > 50%. In their absence, they were < 15%.     

Metal ion-binding ability of tetrapeptides containing alpha-aminoisobutyric acid.

alpha-Aminoisobutyric acid (Aib), one of the Calpha,alpha-disubstituted glycines, is a sterically hindered amino acid that acts as a conformational constraint in peptides. However, studies for the application of the ability of Aib to control conformation are quite few. The paper focuses on the molecular recognition ability of acyclic oligopeptides containing Aib. Liquid-liquid extraction of nine kinds of metal ions from aqueous layers to nonpolar organic layers with acyclic tetrapeptides, X-Trp-Xaa2-Gly-Xaa4-NH-Ar (X = H or C6H5CH2OCO (Z), Xaa2 = Aib or Gly, Xaa4 = Leu or Ala, Ar = phenyl or 3,5-dimethylphenyl) was examined using picrate as the anion of ion pairs. The extraction behaviour of the metal ions with the tetrapeptides was investigated in the pH range from 3 to 9. In the case of basic pH regions, Cu(II) and Ag(I) were effectively extracted with Trp-Aib-Gly-Leu-NH-Ar. Pd(II) was specifically extracted with Trp-Aib-Gly-Leu-NH-Ar in acidic pH regions. The extraction percent (%E) of the peptide host, which has a 3,5-dimethylphenyl group, was even larger than that of the host, which has a phenyl group. Moreover, Pd(II) was extracted with a peptide host which has Leu and a 3,5-dimethylphenyl group in the absence of picrate as the anion of ion pairs. The free alpha-amino group, the turn conformation and the hydrophobicity of peptide molecules were important factors for the extraction of the metals.     

Kinetics and mechanism of peptide synthesis in solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied measuring changes in the fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data couldn't be described by a simple scheme of the second order reaction. The measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: v = kCNaCAEb, where k is the reaction rate constant, CN is the concentration of leucinamide, and LeuNH2, CAE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates. The English version of the paper.     

Important role of the proline residue in the signal sequence that directs the secretion of human lysozyme in Saccharomyces cerevisiae

To elucidate the role of the proline residue in the engineered signal sequence that directs the secretion of human lysozyme in Saccharomyces cerevisiae, we have remodeled an idealized signal sequence L8 = Met-Arg-(Leu)8-Pro-Leu-Ala-Ala-Leu-Gly [Yamamoto, Y., Taniyama, Y., Kikuchi, M., & Ikehara, M. (1987) Biochem. Biophys. Res. Commun. 149, 431-436] in the vicinity of the proline residue. By analyzing the secretory capability of 10 engineered signal sequences, we have shown the following. (1) The proline residue is important for the secretion of human lysozyme and is allowed at position -4, -5, or -6. (2) The secretory capability of the engineered signal sequences is correlated with their predicted conformations. (3) The functional signal sequences that we have investigated can be generalized as follows: Met-Arg-(Leu)n-Pro-(Xaa)-Ala-Leu-Gly where n equals 6-12 and Xaa is Leu, Ala, or Leu-Ala or can be omitted.     

Conformation of two somatostatin analogues in aqueous solution. Study by NMR methods and circular dichroism

Cyclic-disulfide-containing analogues of somatostatin, Xaa1-Cys2-Xaa3-DTrp4-Lys6-Thr5-Xaa7- Xaa8 [Xaa1 = H or DPhe; Xaa3 = Phe or Tyr; Xaa7 = Cys, Me2Cys or Me2DCys; Xaa8 = OH, Thr8 (OH) or Thr8NH2], were examined in aqueous solution by 1H-NMR spectroscopy and circular dichroism. The influence of the helical nature of the disulfide bridge and the presence of exocyclic residues on biological activity were investigated with particular care.     

Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.     

Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs.

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.15 mM) were all approx. 5 mumol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-Sn' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GFR(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, Km: Abu less than Ala less than Pro less than Val less than Ser less than Gly much less than Leu; kcat: Pro greater than Ala greater than Abu greater than Ser greater than Gly much greater than Leu greater than Val; kcat/Km: Abu greater than Pro greater than Ala much greater than Ser greater than Gly = Val much greater than Leu. Km is at a minimum and kcat/Km at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.     

Side chain and backbone dynamics of phospholamban in phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

2H and 15N solid-state NMR spectroscopic techniques were used to investigate both the side chain and backbone dynamics of wild-type phospholamban (WT-PLB) and its phosphorylated form (P-PLB) incorporated into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) phospholipid bilayers. 2H NMR spectra of site-specific CD3-labeled WT-PLB (at Leu51, Ala24, and Ala15) in POPC bilayers were similar under frozen conditions (-25 degrees C). However, significant differences in the line shapes of the 2H NMR spectra were observed in the liquid crystalline phase at and above 0 degrees C. The 2H NMR spectra indicate that Leu51, located toward the lower end of the transmembrane (TM) helix, shows restricted side chain motion, implying that it is embedded inside the POPC lipid bilayer. Additionally, the line shape of the 2H NMR spectrum of CD3-Ala24 reveals more side chain dynamics, indicating that this residue (located in the upper end of the TM helix) has additional backbone and internal side chain motions. 2H NMR spectra of both WT-PLB and P-PLB with CD3-Ala15 exhibit strong isotropic spectral line shapes. The dynamic isotropic nature of the 2H peak can be attributed to side chain and backbone motions to residues located in an aqueous environment outside the membrane. Also, the spectra of 15N-labeled amide WT-PLB at Leu51 and Leu42 residues showed only a single powder pattern component indicating that these two 15N-labeled residues located in the TM helix are motionally restricted at 25 degrees C. Conversely, 15N-labeled amide WT-PLB at Ala11 located in the cytoplasmic domain showed both powder and isotropic components at 25 degrees C. Upon phosphorylation, the mobile component contribution increases at Ala11. The 2H and 15N NMR data indicate significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.     

Importance of the C-terminal phenylalanine of gastrin for binding to the human CCK(2) receptor.

The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.     

An unusual C-H...O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix

An unusual C-terminal conformation has been detected in a synthetic decapeptide designed to analyze the stereochemistry of helix termination in polypeptides. The crystal structure of the decapeptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe reveals a helical segment spanning residues 1-7 and helix termination by formation of a Schellman motif, generated by (D)Ala(8) adopting the left-handed helical (alpha(L)) conformation. The extended conformation at (D)Leu(9) results in a compact folded structure, stabilized by a potentially strong C-H. O hydrogen bond between Ala(4) C(alpha)H and (D)Leu(9) CO. The parameters for C-H. O interaction are Ala(4) C(alpha)H. O=C (D)Leu(9) distance 3.27 A, C(alpha)-H. O angle 176 degrees, and O. H(alpha) distance 2.29 A. This structure suggests that insertion of contiguous D-residues may provide a handle for the generation of designed structures containing more than one helical segment folded in a compact manner.     

Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.     

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees 相似文献   

Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.