scholarly journals The catalytic activity of horse spleen apoferritin. Preliminary kinetic studies and the effect of chemical modification

1973 ◽  
Vol 133 (2) ◽  
pp. 301-309 ◽  
Author(s):  
Charles F. A. Bryce ◽  
R. R. Crichton
Keyword(s):  
Initial Velocity ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Iodoacetic Acid ◽  
Tyrosine Residues ◽  
Progress Curve ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Protein Shell

1. Horse spleen apoferritin catalyses the oxidation of Fe2+ to Fe3+ with molecular O2 as electron acceptor under conditions where a number of other proteins have no such effect. The product is similar to ferritin by a number of criteria. 2. The progress curve is hyperbolic and the increase in initial velocity is linear with increasing apoferritin concentration. With respect to Fe2+ the reaction follows Michaelis–Menten kinetics. The pH-dependence of the reaction was determined between pH4.3 and 6.0. 3. Modification of both tryptophan residues/apoferritin subunit with 2-nitrophenylsulphenyl chloride does not affect either kcat. or Km for the oxidation. Neither does the guanidination of seven out of nine lysine residues/subunit, the modification of nine out of ten arginine residues/subunit with cyclohexanedione, or the nitration of one out of five tyrosine residues/subunit with tetranitromethane. 4. The carboxymethylation of two out of three cysteine residues/subunit and of one out of six histidine residues/subunit can be achieved with iodoacetic acid. This carboxymethylated apoferritin is completely inactive in Fe2+ oxidation. 5. Apoferritin does not take up Fe3+. It appears from these results that Fe2+ is the form in which iron is taken up by ferritin in a reaction where the protein acts as an enzyme which traps the product in the interior of the protein shell.

The reaction of 2,4,6-trinitrobenzene sulfonic acid with pancreatic elastase

1970 ◽  
Vol 48 (11) ◽  
pp. 1249-1259 ◽  
Author(s):  
Leticia Rao ◽  
T. Hofmann
Keyword(s):  
Rate Constant ◽  
Sulfonic Acid ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Trinitrobenzene Sulfonic Acid ◽  
Amino Group ◽  
Tyrosine Residues ◽  
Inactivation Rate Constant ◽  
Lysine Residues ◽  
Ph Range

The reaction of elastase with trinitrobenzene sulfonic acid was investigated in the pH range 9–12. Elastase was found to be inactivated by 2,4,6-trinitrobenzene sulfonic acid. The pH dependence of the pseudo first-order inactivation rate constant showed a pK of 10.3 and gave a Hill plot coefficient of 1.15. Trinitrophenol did not inactivate the enzyme. These results indicate that the inactivation is due to the covalent reaction of trinitrobenzene sulfonic acid with a single group in the enzyme. This group is not the N-terminal since the loss of N-terminal valine was considerably slower than the loss of activity at pH 10.5. The inactivation of elastase with 2,4-dinitrofluorobenzene also showed no correlation with the loss of the N-terminal. When the enzyme was exhaustively treated and fully inactivated with trinitrobenzene sulfonic acid at pH 10.5, the N-terminal valine and two out of three lysine residues were trinitrophenylated. No evidence for the loss of histidine was found. One of the tyrosine residues may be trinitrophenylated as judged from the molar extinction of the trinitrophenylated protein, but it has not been possible to isolate a trinitrophenylated tyrosine-containing peptide. The results can be interpreted in one of two ways: (a) trinitrophenylation of a group with a pK of 10.3, not involved in the activity, inactivates because the introduction of the trinitrophenyl residue causes a denaturation of the enzyme; or (b) a group with a pK of 10.3 controls the active conformation of the enzyme. The results do not exclude the possibility that the N-terminal plays an important role in the activity of the enzyme. Below pH 10.5 the reactivity of the N-terminal is low, indicating that it is buried.At pH 9.0 only the ε-amino group of lysine in position 224 reacted with trinitrobenzene sulfonic acid and full activity was retained. The second-order rate constant for the trinitrophenylation of this group was 25 times higher than that of the ε-amino group of the α-N-benzoyllysine.

scholarly journals Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli

1986 ◽  
Vol 235 (3) ◽  
pp. 651-661 ◽  
Author(s):  
M G P Page ◽  
J P Rosenbusch
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Structural Integrity ◽  
Diazonium Salts ◽  
Tyrosine Residues ◽  
Small Pore ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Pore Forming Proteins ◽  
Small Hydrophobic

The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule.

scholarly journals Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis

1985 ◽  
Vol 232 (1) ◽  
pp. 151-160 ◽  
Author(s):  
G J Hart ◽  
A R Battersby
Keyword(s):  
Euglena Gracilis ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Tyrosine Residues ◽  
Purification And Properties ◽  
Lysine Residues ◽  
Arginine Residues

Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.

scholarly journals Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH

1973 ◽  
Vol 133 (2) ◽  
pp. 289-299 ◽  
Author(s):  
R. R. Crichton ◽  
Charles F. A. Bryce
Keyword(s):  
Conformational Changes ◽  
Difference Spectrum ◽  
Low Ph ◽  
Tryptophan Residue ◽  
Tyrosine Residues ◽  
Ph Values ◽  
Lysine Residues ◽  
Arginine Residues ◽  
The Difference ◽  
Ph Range

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8–10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5–5.0, the subunit reassociates to oligomer in the pH range 3.1–4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.

Review of Progress in Predicting Protein Methylation Sites

2019 ◽  
Vol 23 (15) ◽  
pp. 1663-1670 ◽  
Author(s):  
Chunyan Ao ◽  
Shunshan Jin ◽  
Yuan Lin ◽  
Quan Zou
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Protein Methylation ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Regulatory Enzymes ◽  
Lysine Residues ◽  
Arginine Residues

Protein methylation is an important and reversible post-translational modification that regulates many biological processes in cells. It occurs mainly on lysine and arginine residues and involves many important biological processes, including transcriptional activity, signal transduction, and the regulation of gene expression. Protein methylation and its regulatory enzymes are related to a variety of human diseases, so improved identification of methylation sites is useful for designing drugs for a variety of related diseases. In this review, we systematically summarize and analyze the tools used for the prediction of protein methylation sites on arginine and lysine residues over the last decade.

scholarly journals Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies

1982 ◽  
Vol 205 (2) ◽  
pp. 361-372 ◽  
Author(s):  
E J Emanuel ◽  
A D Brampton ◽  
D R Burton ◽  
R A Dwek
Keyword(s):  
Immunoglobulin G ◽  
Water Soluble ◽  
Small Peptide ◽  
Experimental Conditions ◽  
Salt Ions ◽  
Salt Range ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Equilibrium Process ◽  
Glycine Ethyl Ester

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q-immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15-0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9-12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338-344].

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide

1977 ◽  
Vol 55 (4) ◽  
pp. 424-432
Author(s):  
Bernard R. Glick ◽  
Lewis J. Brubacher
Keyword(s):  
Kinetic Studies ◽  
Peptide Inhibitors ◽  
Tryptophan Residue ◽  
Difference Spectroscopy ◽  
Tyrosine Residues ◽  
Residue Determination ◽  
Chain Cleavage ◽  
Modified Enzyme ◽  
Kinetics Of ◽  
Twofold Decrease

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine, (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH 6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate α-N-benzoyl-L-arginine ethyl ester were studied in the region of pH 3.5–9.0. All substrates (and inhibitors) tested, with the exception of α-N-benzoyl-L-arginine p-nitroanilide, displayed approximately a twofold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is modified is probably Trp-177.

Influence on drug efficacy of the binding behavior of pioglitazone hydrochloride with tryptophan residues, and tyrosine residues in bovine transferrin

2018 ◽  
Vol 51 (10) ◽  
pp. 554-562
Author(s):  
Bao-Sheng Liu ◽  
Chun-Dan Wang ◽  
Gang Bian ◽  
Li-Hua Ma ◽  
Hong-Cai Zhang ◽  
...  
Keyword(s):  
Drug Efficacy ◽  
Tyrosine Residues ◽  
Binding Behavior ◽  
Tryptophan Residues ◽  
Bovine Transferrin

scholarly journals Evading the Proteasome: Absence of Lysine Residues Contributes to Pertussis Toxin Activity by Evasion of Proteasome Degradation

2007 ◽  
Vol 75 (6) ◽  
pp. 2946-2953 ◽  
Author(s):  
Zoë E. V. Worthington ◽  
Nicholas H. Carbonetti
Keyword(s):  
Pertussis Toxin ◽  
Intracellular Transport ◽  
Cellular Activity ◽  
Wild Type ◽  
Proteasome Degradation ◽  
Lysine Residues ◽  
Arginine Residues ◽  
A Subunit ◽  
Adp Ribosyltransferase

ABSTRACT Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.

scholarly journals Anomalous pH-dependence of the activity of human matrilysin (matrix metalloproteinase-7) as revealed by nitration and amination of its tyrosine residues

2005 ◽  
Vol 386 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Yuko MUTA ◽  
Hiroshi ONEDA ◽  
Kuniyo INOUYE
Keyword(s):  
Matrix Metalloproteinase ◽  
Strong Influence ◽  
Negative Charge ◽  
Ph Dependence ◽  
Michaelis Constant ◽  
Pka Values ◽  
Tyrosine Residues ◽  
Alkaline Side ◽  
Matrix Metalloproteinase 7

Matrilysin activity exhibits a broad bell-shaped pH-dependence profile, with pKa values of 4.0 and 9.8. A maximum of five out of eight tyrosine residues in matrilysin were nitrated with tetranitromethane. On nitration of between one and five tyrosines, pKa at the alkaline side (pKe2) was shifted from 9.8 to 10.3–10.6, while that at the acidic side (pKe1) was not altered. The pKe2 that was shifted by nitration to 10.3–10.6 was restored to 9.4–9.7 by subsequent amination, suggesting that the shift in pKe2 is induced by a negative charge introduced on the most reactive tyrosine, Tyr-150. The Michaelis constant (Km) observed at pH 10 was decreased by nitration as a result of the increase in pKe2, suggesting that the residue with pKe2 may play a role in the recognition of substrate. When four or five tyrosines were nitrated, the activity at pH <7 decreased significantly, while that at pH 7–10 was unchanged, and thus the pH-dependence was not bell-shaped, but anomalous, with a third pKa (pKe3) of 6.2–6.4 in addition to pKe1 and pKe2. This suggests the possibility that a newly introduced nitrotyrosine residue has a strong influence on the activity as an ionizable group.

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