The Reaction of Chymotrypsin with 2,3-Butanedione Trimer

1975 ◽  
Vol 53 (3) ◽  
pp. 275-283 ◽  
Author(s):  
H. Fliss ◽  
N. M. Tozer ◽  
T. Viswanatha
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Residual Activity ◽  
Amino Acid Residues ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Chymotrypsin A

A method for the preparation of the trimer of 2,3-butanedione has been developed. The reaction of this trimer with chymotrypsin Aα was examined in the presence or absence of light. Under conditions of exclusion of light, modification of one to two arginine residues and of a similar number of lysine residues could be achieved without any loss of enzymatic activity. The trimer facilitated a rapid photoinactivation of the enzyme with little or no modification of the above amino acid residues. Such photoinactivation was not promoted by the monomer 2,3-butanedione. Enzyme irradiated in the presence of the trimer was found to react with proflavine and diisopropylfluorophosphate to an extent greater than that expected on the basis of residual activity present. Proflavine protected the enzyme from the trimer promoted photoinactivation.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

2010 ◽  
Vol 432 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Emily R. Slepkov ◽  
Alan Pavinski Bitar ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Phospholipase C ◽  
Bacterial Pathogen ◽  
Amino Acid Residues ◽  
C Terminus ◽  
N Terminus ◽  
Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

scholarly journals The N-terminal amino acid sequence of pig kidney endopeptidase-24.11 shows homology with pro-sucrase-isomaltase

1986 ◽  
Vol 240 (1) ◽  
pp. 305-308 ◽  
Author(s):  
I S Fulcher ◽  
D J Pappin ◽  
A J Kenny
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Consensus Sequence ◽  
Terminal Segment ◽  
Amino Acid Residues ◽  
Endopeptidase 24.11 ◽  
Cytoplasmic Face ◽  
Arginine Residues ◽  
Terminal Amino ◽  
Hydrophobic Anchor

Endopeptidase-24.11 (EC 3.4.24.11), a widely distributed ectoenzyme, was isolated from pig kidneys by detergent solubilization of membranes and immuno-affinity chromatography. In all, 12 preparations of the enzyme were submitted to solid-phase sequencing, yielding a consensus sequence of 25 amino acid residues of the N-terminal segment. Some samples were treated with either trypsin or Staphylococcus aureus V8 proteinase before sequencing. There were four lysine and one arginine residues in the first nine positions. This segment was susceptible to hydrolysis by trypsin and, in some samples, to endogenous proteinases. From residue 19 onwards, the sequence became intensely hydrophobic. There was a striking homology with the N-terminal sequence of pro-sucrase-isomaltase. From Lys7 to Leu20 there were seven identical amino acid residues and four conservative substitutions. We suggest that endopeptidase-24.11 is topologically similar to this glycosidase, the N-terminus at the cytoplasmic face and hydrophobic segment serving the roles of both signal peptide and hydrophobic anchor.

scholarly journals Surface-exposed Amino Acid Residues of HPV16 L1 Protein Mediating Interaction with Cell Surface Heparan Sulfate

2007 ◽  
Vol 282 (38) ◽  
pp. 27913-27922 ◽  
Author(s):  
Maren Knappe ◽  
Sabrina Bodevin ◽  
Hans-Christoph Selinka ◽  
Dorothe Spillmann ◽  
Rolf E. Streeck ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Capsid Protein ◽  
Mutational Analysis ◽  
Cell Attachment ◽  
Human Papillomaviruses ◽  
Amino Acid Residues ◽  
Cell Binding ◽  
Lysine Residues

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.

scholarly journals The amino acid sequence of sesame (Sesamum indicum L.) and castor (Ricinus communis L.) cytochrome c

1971 ◽  
Vol 121 (3) ◽  
pp. 439-446 ◽  
Author(s):  
E. W. Thompson ◽  
M. Richardson ◽  
D. Boulter
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Mung Bean ◽  
Ricinus Communis ◽  
Sesamum Indicum ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Ricinus Communis L ◽  
Lysine Residues

The amino acid sequences of sesame (Sesamum indicum L.) and castor (Ricinus communis L.) cytochrome c were determined by using 1.5μmol of protein from each species. Both molecules consist of a single chain of 111 amino acid residues and are homologous with other mitochondrial cytochrome c molecules. Both have an N-acetylated ‘tail’ of eight amino acids and two ∈-N-trimethyl-lysine residues, as also reported for wheat germ (Delange, Glazer & Smith, 1969) and mung-bean cytochrome c (Thompson, Laycock, Ramshaw & Boulter, 1970). Two different preparations of castor cytochrome c differed by one residue. This was glutamic acid for glutamine in position 100. The results for sesame and castor cytochrome c led to a re-examination and subsequent correction to the N-terminal region of the mung-bean cytochrome c sequence, as given by Thompson et al. (1970).

scholarly journals Structural studies on wheat (Triticum aestivum) proteins lacking phenylalanine and histidine residues

1975 ◽  
Vol 149 (3) ◽  
pp. 725-732 ◽  
Author(s):  
D G Redman
Keyword(s):  
Amino Acids ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
Structural Studies ◽  
Amino Acid Residues ◽  
Sequencing Studies ◽  
Arginine Residues ◽  
Terminal Amino ◽  
Methionine Residues ◽  
High Degree

1. Three very similar proteins, each of approx. 120 amino acid residues but lacking phenylalanine and histidine, were isolated from wheat (Triticum aestivum) flour in sufficient quantities for further structural studies. 2. Each protein, after reduction and carboxymethylation, was cleaved at the three methionine residues with CNBr to give four major peptides, which were isolated. These peptides are suitable for future sequencing studies, as the sums of their amino acid compositions are in good agreement with those of the whole proteins. 3. The N- and C-terminal peptides were identified. 4. Evidence from amino acid analyses, N-terminal amino acids and electrophoretic mobilities of the peptides suggests a high degree of homology between the proteins. Definite differences in C-terminal amino acids and the number of glycine, alanine and arginine residues were found in the C-terminal peptides.

scholarly journals Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases

1992 ◽  
Vol 286 (3) ◽  
pp. 923-927 ◽  
Author(s):  
R Myllylä ◽  
V Günzler ◽  
K I Kivirikko ◽  
D D Kaska
Keyword(s):  
Amino Acid ◽  
Reaction Mechanisms ◽  
Catalytic Site ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Peptide Substrate ◽  
Histidine Residues ◽  
Lysyl Hydroxylase ◽  
Diethyl Pyrocarbonate ◽  
Lysine Residues

A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.

scholarly journals The primary structure of histone H2A from the nematode Caenorhabditis elegans

1987 ◽  
Vol 243 (1) ◽  
pp. 297-300 ◽  
Author(s):  
J R Vanfleteren ◽  
S M Van Bun ◽  
J J Van Beeumen
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Primary Structure ◽  
Amino Acid Residues ◽  
Histone H2a ◽  
Nematode Caenorhabditis Elegans ◽  
Calf Thymus ◽  
N Terminus ◽  
Lysine Residues ◽  
Complete Primary Structure

The complete primary structure of histone H2A from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 126 amino acid residues and has a blocked N-terminus. By comparison with calf thymus histone H2A, the nematode protein shows five deletions, two insertions and 16 substitutions. Most of the changes occur in the N- and C-terminal regions of the molecule, whereas the central part covering the residues 21-120 is quite well conserved. The lysine residues 5, 8 and 10 were found to be partially acetylated.

scholarly journals Evidence for essential histidine and dicarboxylic amino-acid residues in the active site of UDP-glucose : solasodine glucosyltransferase from eggplant leaves.

2003 ◽  
Vol 50 (2) ◽  
pp. 567-572 ◽  
Author(s):  
Paulina Nawłoka ◽  
Małgorzata Kalinowska ◽  
Cezary Paczkowski ◽  
Zdzisław A Wojciechowski
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Active Site ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Chemical Probes ◽  
Dicarboxylic Amino Acid ◽  
Enzyme Substrates ◽  
Arginine Residues

Effects of several chemical probes selectively modifying various amino-acid residues on the activity of UDP-glucose : solasodine glucosyltransferase from eggplant leaves was studied. It was shown that diethylpyrocarbonate (DEPC), a specific modifier of histidine residues, was strongly inhibitory. However, in the presence of excessive amounts of the enzyme substrates, i.e. either UDP-glucose or solasodine, the inhibitory effect of DEPC was much weaker indicating that histidine (or histidines) are present in the active site of the enzyme. Our results suggest also that unmodified residues of glutamic (or aspartic) acid, lysine, cysteine, tyrosine and tryptophan are necessary for full activity of the enzyme. Reagents modifying serine and arginine residues have no effect on the enzyme activity.

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