Leucyl Residues Research Articles

Refined 2 Å X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase: Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4 12 12 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure. Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined. A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen & Bode. 1983). The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen & Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.

ChemInform Abstract: RENIN INHIBITORS. SUBSTITUTION OF THE LEUCYL RESIDUES OF LEU‐LEU‐VAL‐PHE‐OCH3 WITH 3‐AMINO‐2‐HYDROXY‐5‐METHYLHEXANOIC ACID

AbstractDas L‐Leucinderivat (I) ergibt über das Säurechlorid den Aldehyd (II) und weiter die Additionsverbindung (III), die zu einem Diastereomeren‐Gemisch der Carbonsäuren (IV) führt.

Synthesis and characterization of neurotensin analogues for structure/activity relationship studies. Acetyl-neurotensin-(8--13) is the shortest analogue with full binding and pharmacological activities.

Neurotensin and several sequence analogues have been synthesized using solid‐phase technology. The purity of the following derivatives: neurotensin, neurotensin‐(10–13), neurotensin‐(9–13). neurotensin‐(8–13), neurotensin‐(6–13), neurotensin‐(4–13), [Cit8]neurotensin‐(8–13), [Lys8]neurotensin‐(8–13), [Cit9]neurotensin‐(8–13),[Lys9]neurotensin‐(8–13), [Phe11]neurotensin‐(8–13), [Ala12]neurotensin‐(8–13) and [Ala13]‐ neurotensin‐(8–13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse‐phase high‐ performance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N‐acetylation of neurotensin‐(6–13), neurotensin‐(8–13), [Cit8]neurotensin‐(8–13), [Cit9]‐ neurotensin‐(8–13), [Lys8]neurotensin‐(8–13), [Lys9]neurotensin‐(8–13) and [Phe11]neurotensin‐(8–13), as well as native xenopsin, were all tested for binding competition with [3H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of HT 29 cells. In addition to these radioreceptor assays on neural and extraneural targets, a pharmacological test (contraction of guinea pig ileum in the presence of neostigmine) was used to compare the behavior of the synthetic analogues. The use of these three biological systems enabled us to obtain consistent results. A good parallel was observed between the degree of fixation and pharmacological effects for entire neurotensin and for C‐terminal region analogues up to the size of neurotensin‐ (8–13). The two peptides neurotensin‐(6‐ 13) and neurotensin‐(4–13) had an abnormally high affinity for rat brain synaptic membrane binding sites compared to a relatively low contracting activity. The C‐terminal peptide ‐Arg‐Arg‐Pro‐Tyr‐Ile‐Leu fulfills all the structural requirements for mimicking the entire sequence, provided its α‐amino end is protected by acetylation. The guanidinium structure of residues 8 and 9 are not of vital importance, since they could be efficiently replaced by amino groups of lysyl side chains. Xenopsin, which can be considered as a natural analogue of neurotensin‐(8–13), acts similarly to acetyl‐neurotensin‐(8–13). Removal of the phenolic function of residue 11 induces a decrease in neurotensin effects. The C‐terminal isoleucyl and leucyl residues could not be replaced by alanine without complete loss of the three activities tested.

Renin inhibitors. Substitution of the leucyl residues of Leu-Leu-Val-Phe-OCH3 with 3-amino-2-hydroxy-5-methylhexanoic acid.

The 2S,3S and 2R,3S diastereoisomers of the hydroxy amino acid 3-amino-2-hydroxy-5-methylhexanoic acid (AHMHA) were synthesized and substituted for the leucyl residues of Leu-Leu-Val-Phe-OCH3 to yield the following analogues: AHMHA-Leu-Val-Phe-OCH3, AHMHA-Val-Phe-OCH3, and Leu-AHMHA-Val-Phe-OCH3. These analogues were tested in vitro for their ability to inhibit human amniotic renin. All of the analogues were found to inhibit renin to some extent with inhibitory constants in the range of 10(-3) to 10(-4) M. The analogues AHMHA-Leu-Val-Phe-OCH3 and AHMHA-Val-Phe-OCH3 exhibited competitive inhibition when the 2S,3S isomer of AHMHA was employed and noncompetitive kinetics when the 2R,3S isomer of AHMHA was used. For the Leu-AHMHA-Val-Phe-OCH3 analogues, competitive kinetics were observed regardless of the isomer of AHMHA employed. These latter analogues also proved to be the most active in the above series.

Hemoglobin Great Lakes (β68 [E12] Leucine → Histidine): A New High-Affinity Hemoglobin

Abstract Hemoglobin Great Lakes, beta 68 (E12) Leu replaced by His is a new high oxygen affinity hemoglobin variant discovered in a 29-yr-old female having numerous hospitalizations for thrombophlebitis associated with mild erythrocytosis. The mutant hemoglobin has normal stability and normal electrophoretic mobility, but increased oxygen affinity (P-50 16.1 mm Hg at 37 degrees C, pH 7.4) and reduced cooperativity. The abnormal beta-chain could be separated on globin chain chromatography on carboxymethyl/cellulose in spite of the normal electrophoretic mobility of the intact hemoglobin. The leucyl residue at beta 68th position (E12) is in the middle of E-helix, which is part of the heme pocket and next to the valine (E11), which is the heme binding site. The substitution of proline for leucine in hemoglobin Mizuho resulted in the distortion of tertiary structure of the beta-chains and lead to a serious instability of hemoglobin molecule. However, the substitution of this residue by histidine in hemoglobin Great Lakes is not associated with hemoglobin instability.

Hemoglobin Great Lakes (beta 68 [E12] leucine replaced by histidine): a new high-affinity hemoglobin

Hemoglobin Great Lakes, beta 68 (E12) Leu replaced by His is a new high oxygen affinity hemoglobin variant discovered in a 29-yr-old female having numerous hospitalizations for thrombophlebitis associated with mild erythrocytosis. The mutant hemoglobin has normal stability and normal electrophoretic mobility, but increased oxygen affinity (P-50 16.1 mm Hg at 37 degrees C, pH 7.4) and reduced cooperativity. The abnormal beta-chain could be separated on globin chain chromatography on carboxymethyl/cellulose in spite of the normal electrophoretic mobility of the intact hemoglobin. The leucyl residue at beta 68th position (E12) is in the middle of E-helix, which is part of the heme pocket and next to the valine (E11), which is the heme binding site. The substitution of proline for leucine in hemoglobin Mizuho resulted in the distortion of tertiary structure of the beta-chains and lead to a serious instability of hemoglobin molecule. However, the substitution of this residue by histidine in hemoglobin Great Lakes is not associated with hemoglobin instability.

Critical chain length for the formation of α- and β-polypeptide conformations in the solid state. Syntheses and conformations of oligopeptides with alternateL-alanyl andL-leucyl residues

A series of oligopeptides with alternate L-alanyl and L-leucyl residues Nps-(L-Leu-L-Ala)n-OEt and Nps-L-Ala(L-Leu-L-Ala)m-OEt (n= 1–6 and 8, and m= 1–5) have been prepared by stepwisesynthesis with o-nitrophenylsulphenyl N-carboxy-α-amino-acid anhydrides for lower oligopeptides and fragment condensation with N-hydroxysuccinimide and dicyclohexylcarbodi-imide for higher oligopeptides; assignment of conformation to the products in the solid state has been made on the basis of i.r. and for i.r. spectroscopic results and X-ray diffraction measurements. α-Helical conformations have been found in deca- and higher peptides. The β-structure, on the other hand, appears to begin at the heptapeptide level.

ChemInform Abstract: STUDIES OF PEPTIDE ANTIBIOTICS. XXXVII. SYNTHESIS OF (1,1′,3,3′‐L‐ALANINE)‐GRAMICIDIN S

AbstractKondensation der Peptidderivate (I) und (II), jeweils nach üblichen Methoden durch schrittweisen Aufbau dargestellt, gibt das Decapeptidderivat (IIIa), das über die Stufen (IIIb) und (IIIc) in das Cyclopeptid (IVa) übergeführt wird (Ausb. bezogen auf (IIIa)).

Studies of Peptide Antibiotics. XXXVII. Synthesis of [1,1′,3,3′-l-Alanine]-Gramicidin S

Abstract An analog containing alanyl residues in place of valyl and leucyl residues in gramicidin S, namely [1,1′,3,3′-l-alanine]-gramicidin S, was synthesized to investigate the influence of the bulky side chains of the amino acid residues for antibacterial activity and conformation. The analog has the small aliphatic side chains of alanyl residues on one side of the ring frame in the gramicidin S-structure. In the experiments of optical rotatory dispersion and circular dichroism, this analog gave curves similar in shape to these of gramicidin S, and thus should have a similar conformation to that of gramicidin S. The analog showed, however, no antibacterial activity. Thus the four methyl side chains of alanyl residues in the analog are not sufficient for the substance to exhibit biological activity.

Amino acid sequence of the small core protein from bacteriophage phiX174.

The amino acid sequence of small core protein of bacteriophage phiX174 has been determined by a combination of automated Edman degradation of the intact polypeptide and by analysis of tryptic and thermolytic peptides. The six lysyl and six arginyl residues of this 37-residue polypeptide are concentrated in two structurally homologous 12-residue segments of the sequence. The hydrophobic residues of valine, tryptophan, tyrosine, and phenylalanine are contained in the carboxyl-terminal nine residues of the protein, together with one of the two leucyl residues and two of the three glutaminyl residues. The single free carboxyl group in the protein is the alpha-COOH of the C-terminal phenylalanyl residue. The overall sequence of this small core protein suggests that it may function as a DNA-condensing protein. The protein sequence presented here corresponds exactly to the DNA base sequence of the cistron J region of the phiX174 genome determined in another laboratory.

Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen.

The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.

The Influence of Glycyl Residues on the Flexibility of Peptide Hormones in Solution. A 13C-Nuclear-Magnetic-Resonance Study of Luteinizing Hormone-Releasing Hormone (Luliberin) and Its Des-glycinamide10 N-ethylamide Analog

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.

Neurotensin: specific binding to synaptic membranes from rat brain.

The binding of neurotensin to synaptic membranes from rat brain was studied at 24 degrees with the use of [3H]neurotensin. The binding was found to be highly specific, saturable, and reversible. Values for KD of 2 nM and 0.9 nM were derived from equilibrium and kinetic experiments, respectively. Virtually no degradation of neurotensin was observed in the incubation medium after exposure to synaptic membranes under the conditions of the binding studies. Competitive inhibition of [3H]neurotensin binding by partial sequences of neurotensin revealed that the addition of the residue arginine-8 to the neurotensin-(9-13)-pentapeptide increases about 500-fold the relative binding potency, whereas the remaining portion of the NH2-terminal region is mainly responsible for full pharmacological potency; the COOH-terminal leucyl residue is essential for binding.

Structural evidence for γ globin chain synthesis in adult bone marrow cultures

Summary In adult human bone marrow cultures increased amounts of a protein with chromatographic characteristics of fetal hemoglobin are synthesized. The structural similarity between this ( 3 H) leucyl labelled protein and fetal Hb was examined. Upon partial sequence of the ( 3 H) labelled protein and carrier γ globin, incorporation of radioactivity was found only at positions normally occupied by leucyl residues in the γ chain. There was complete coincidence of radioactivity and optical density profiles of Sephadex G-50 chromatography of cyanogen bromide fragments of the carrier γ globin and the ( 3 H) labelled protein produced in culture. Specific activities of cyanogen bromide fragments were as expected by their composition in leucyl residues. Thus, by all chromatographic and structural criteria, the labelled protein appears to the human γ chain.

Influence of D and L amino‐acid residues on the conformation of peptides in solution: A carbon‐13 nuclear magnetic resonance study of cyclo(prolyl‐leucyl)

AbstractThe 13C chemical shifts and spin‐lattice relaxation times are reported for cyclo(L‐Pro‐L‐Leu) and cyclo(L‐Pro‐D‐Leu). The chemical shifts of the D and L leucyl residues in the cyclic peptides differ from each other by 1.8 and 3.6 parts per million for the α and β carbons, respectively. The α‐carbons of the prolyl residues differ by 1.0 ppm as a consequence of proximity to a D or an L leucyl residue. The 13C spin‐lattic relaxation time(T1) of the prolyl residues, but not the leucyl residues, in both compounds are indicative of difference in conformational equilibria within the pyrrolidine ring in the L‐L isomer as compared to the L‐D isomer. Anisotropic overall molecular reorientation is not responsible for the differences observed in the T1 values. The differences in T1 values and chemical shifts between cyclo(L‐Pro‐L‐Leu) and cyclo(L‐Pro‐D‐Leu) appear to result from a difference in conformations of the two diketopiperazine rings.

Sequence peptide polymers: 3. Synthesis and conformational study in solution of poly ( [formula omitted

The synthesis and characterization of poly[Leu-Leu-(ε-Z)-Lys] and its conformational study in organic solvents are reported. The sequential polypeptide has been obtained with a satisfactory optical purity by the self-condensation method of the p-nitrophenyl ester of the corresponding tripeptide derivative according to the general procedure of De Tar et al. Poly[Leu-Leu-(ε-Z)-Lys] showed a right-handed α-helical conformation in solution of helicogenic solvents, as displayed by circular dichroism measurements and ultra-violet absorption spectra. The presence of the α-helix has been also detected in the solid state by infra-red spectroscopy. The helix stability of the polypeptide in chloroform—dichloroacetic acid solution is considerably high. This property may be related to the ordered sequence of the bulky leucyl residues which give rise to a screening effect, protecting the chain backbone from the acid solvent molecules.

Synthesis and properties of Clostridium acidi-urici (Leu2)-ferredoxin: a function of the peptide chain and evidence against the direct role of the aromatic residues in electron transfer.

Tyrosyl or other aromatic residues generally occur in two conserved positions in the peptide chain of clostridial-type ferredoxins and have been implicated in the electron transfer function of these iron-sulfur proteins. We have prepared and determined some of the properties of a derivative of Clostridium acidi-urici ferredoxin, [Leu(2)]-ferredoxin, in which a leucyl residue has been substituted for the tyrosyl residue in position 2 from the amino terminus. [Leu(2)]-ferredoxin is fully active as an electron carrier in two biological assays, the phosphoroclastic enzyme system and the ferredoxin-dependent reduction of cytochrome c in the presence of ferredoxin-TPN reductase and TPNH. Quantitative electron paramagnetic resonance experiments indicate that [Leu(2)]-ferredoxin accepts nearly two electrons upon enzymatic reduction by pyruvate-ferredoxin oxidoreductase and an excess of pyruvate. If electron transfer to an iron-sulfur cluster is the rate-limiting step in the assays used, and if the rate of electron transfer through Tyr(30) is not much faster than through Tyr(2), these results indicate that the primary pathway of electron transfer in clostridial-type ferredoxins is not via Tyr or other aromatic amino-acid residues. The syntheses of other ferredoxin derivatives with amino-acid substitutions or deletions in positions 1 and 2 indicate that a large bulky residue, but not necessarily an aromatic residue, is needed in position 2 for the stability of this ferredoxin. The residue in position 2, therefore, appears to act as a hydrophobic shield for an iron-sulfur cluster.

Hb-Genova ( α2β228(bio)leu→pro); methods for detection and analysis of unstable hemoglobins

The third occurrence of the unstable β chain variant Hb-Genova is described. The variant is characterized by replacement of a leucyl residue in position 28(B 10) by a prolyl residue. The abnormality was discovered in a Canadian woman of Ukrainian ancestry, who had a persistent anemia with Heinz bodies prevalent since early childhood. The techniques used in the detection of unstable hemoglobins are reviewed.

Syntheses and enzymic hydroxylation of protocollagen model peptides containing glutamyl or leucyl residue

Syntheses and enzymic hydroxylation of protocollagen model peptides containing glutamyl or leucyl residue

Conformational studies on copolymers of L-lysine and L-leucine: circular dichroism and potentiometric titration studies.

AbstractConformational aspects of a series of copolymers of L‐Leucine and L‐leucine [poly‐(LysxLeuy)] containing 0 to 0.41 mole fraction L‐leucine have been studied by circular dichroism (CD) and potentiometric titration in 0.05M KF solution. CD studies on the α‐helical conformation showed a dependence of the magnitude of the CD ellipticity band at 222 nm on copolymer composition; the [θ]222 decreasing with higher leucine contents. This was interpreted as the result of an increase of the hydrophobicity of the environment of the amide group due to the presence of the leucyl residues. Values of the Zimm‐Rice parameter, σ, for the copolymers were obtained from the potentiometric titrations and used to fit theoretical curves to the experimental data. Using the variation of σ with polymer composition, a value of σ for the leucyl residue was estimated to be 6.3 × 10−2, assuming independence of σ on the amino acid sequence in the copolymer. The free energy change for the conversion of one mole residue from uncharged helix to uncharged coil, ΔGhc°, was also obtained from the titration data for each copolymer up to a leucine mole fraction of 0.16; a value of 385 cal mole−1 was estimated for ΔGhc° for a leucyl residue. These values for σ and ΔGhc° are compared with other values in the literature for various amino acid residues obtained from titration and melting curve data.