The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

The effects of hydrophobic mismatch between phosphatidylcholine bilayers and transmembrane alpha-helical peptides depend on the nature of interfacially exposed aromatic and charged residues

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects

Understanding the hydrophilicity/hydrophobicity of amino acid side chains in peptides/proteins is one the most important aspects of biology. Though many hydrophilicity/hydrophobicity scales have been generated, an "intrinsic" scale has yet to be achieved. "Intrinsic" implies the maximum possible hydrophilicity/hydrophobicity of side chains in the absence of nearest-neighbor or conformational effects that would decrease the full expression of the side-chain hydrophilicity/hydrophobicity when the side chain is in a polypeptide chain. Such a scale is the fundamental starting point for determining the parameters that affect side-chain hydrophobicity and for quantifying such effects in peptides and proteins. A 10-residue peptide sequence, Ac-X-G-A-K-G-A-G-V-G-L-amide, was designed to enable the determination of the intrinsic values, where position X was substituted by all 20 naturally occurring amino acids and norvaline, norleucine, and ornithine. The coefficients were determined by reversed-phase high-performance liquid chromatography using six different mobile phase conditions involving different pH values (2, 5, and 7), ion-pairing reagents, and the presence and absence of different salts. The results show that the intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides (proteins) is independent of pH, buffer conditions, or whether C(8) or C(18) reversed-phase columns were used for 17 side chains (Gly, Ala, Cys, Pro, Val, nVal, Leu, nLeu, Ile, Met, Tyr, Phe, Trp, Ser, Thr, Asn, and Gln) and dependent on pH and buffer conditions, including the type of salt or ion-pairing reagent for potentially charged side chains (Orn, Lys, His, Arg, Asp, and Glu).     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

Kinetic analysis of the role of histidine chloramines in hypochlorous acid mediated protein oxidation

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and chloride ions by the heme enzyme myeloperoxidase (MPO) released from activated leukocytes. In addition to its potent antibacterial effects, excessive HOCl production can lead to host tissue damage, with this implicated in human diseases such as atherosclerosis, cystic fibrosis, and arthritis. HOCl reacts rapidly with biological materials, with proteins being major targets. Chlorinated amines (chloramines) formed from Lys and His side chains and alpha-amino groups on proteins are major products of these reactions; these materials are however also oxidants and can undergo further reactions. In this study, the kinetics of reaction of His side-chain chloramines with other protein components have been investigated by UV/visible spectroscopy and stopped flow methods at pH 7.4 and 22 degrees C, using the chloramines of the model compound 4-imidazoleacetic acid and N-alpha-acetyl-histidine. The second-order rate constants decrease in a similar order (Cys > Met > disulfide bonds > Trp approximately alpha-amino > Lys > Tyr > backbone amides > Arg) to the corresponding reactions of HOCl, but are typically 5-25 times slower. These rate constants are consistent with His side-chain chloramines being important secondary oxidants in HOCl-mediated damage. These studies suggest that formation and subsequent reactions of His side-chain chloramines may be responsible for the targeted secondary modification of selected protein residues by HOCl that has previously been observed experimentally and highlight the importance of chloramine structure on their subsequent reactivity.     

Side-chain flexibility in proteins upon ligand binding

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino-acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo-protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets.     

Porcine spleen cathepsin H hydrolyzes oligopeptides solely by aminopeptidase activity

Cathepsin H purified from porcine spleens was studied for its specificity against various peptide and denatured protein substrates. The enzyme degraded all peptide substrates exclusively by an aminopeptidase activity. The enzyme preferentially released NH2-terminal amino acid residues with large hydrophobic (Phe, Trp, Leu, and Tyr) or basic (Arg and Lys) side chains. Amino acids containing small or polar side chains were not released. Peptides with a proline in the NH2-terminal or penultimate positions were not hydrolyzed either. Large polypeptides such as reduced and carboxymethylated soybean trypsin inhibitor and aldolase were not degraded. These results indicate that cathepsin H is an exopeptidase but not an endopeptidase. We propose that the biological role of this enzyme is the degradation of tissue proteins in lysosomes by its aminopeptidase activity.     

Lysozyme Modification by the Fenton Reaction and Gamma Radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H 2 O 2 ) or hydroxyl radicals produced by &#110 radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. &#110 radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met=Cys > Lys > Ile+Leu > Gly > Pro=Phe>Thr+Ala>Trp=Ser>Arg>Asp+Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met >His > Ile+Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by &#110 radiation.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

Modified Proteases for Peptide Synthesis in Organic Media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.     

Modified proteinases in peptide synthesis in organic media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.     

Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.