Effect of N-bromosuccinimide-modification of tyrosine side chains of cardiotoxin II of the Indian cobra on biological activity

The essential role of tyrosine residue(s) of cardiotoxin II in the biological activity of the toxin was evaluated using N-bromosuccinimide. N-bromosuccinimide effected oxidation of the tyrosine residues in cardiotoxin II with enhancement in absorbance at 260 nm. The influence of various solvent media such as acetate-formate buffer (pH 4.0), 0.01 N H₂SO₄ (pH 2.0) and Tris-HCl buffer (pH 8.5) on oxidation of tyrosine residues was exa mined. In comparison with 0.01 N H₂S O₄, acetate-formate buffer could prevent secondary oxidations as revealed by lower consumption of oxidant, N-bromosuccinimide, to achieve oxidation. In Tris-HCl buffer oxidation of tyrosine did not take place effectively. N-iodo-succinimide caused only limited oxidation as evident from minor increase in absorbance at 260 nm. N-chlorosuccinimide was completely ineffective. Oxidation of cardiotoxin II with 3.75 equivalents of N-bromosuccinimide tyrosine residue led to complete loss of lethal activity. However, the derivative retained the ability to protect bacterial protoplasts from lysis in solutions of low tonicity. Unlike cardiotoxin II oxidized with N-chlorosuccinimide (50 equivalents/mol of toxin) which retained lethal activity as well as the ability to protect protoplasts from lysis, performic acid-oxidized toxin had lost both the activities.     

Tryptophan residue essential for activity of Naja naja atra phospholipase A2

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.     

2-D-NMR study of the conformation characteristics of toxin 3 from Naja naja siamensis venom

The spatial structure of "long" toxin 3 Naja naja siamensis in solution has been studied by methods of two-dimensional (2D) 1H NMR spectroscopy. The individual signal assignments for 67 out of 71 residues and analysis of nuclear Overhauser effects between distinct protons of the molecule allowed the comparison of the toxin 3 conformations at different pH values and temperatures. It was shown that the deprotonated imidazole ring of His22 residue (at pH greater than or equal to 7,5) is surrounded by the side chains of Cys17, Pro18, Val23, Cys24, Cys45, Ala46 and Thr48 residues. On the contrary, the protonated imidazole ring of His22 (at pH less than 4,0) is exposed into solvent. Ionization of His22 is accompanied by a change in the Tyr25 aromatic ring orientation and affects the conformational mobility of the Cys17, His22, Cys45 and Ala47 side chains. The revealed conformational features of toxin 3 in solution are discussed in connection with the differences between "long" and "short" neurotoxins in the kinetics of their binding to acetylcholine receptor.     

Hemin-H2O2-NO2(-) induced protein oxidation and tyrosine nitration are different from those of SIN-1: a study on glutamate dehydrogenase nitrative/oxidative modification

Protein oxidation and tyrosine nitration are two major post-translational modifications of protein by reactive nitrogen oxide species, which are mainly produced by peroxynitrite and heme peroxidases (hemin)-H(2)O(2)-NO(2)(-) system. We report herein some novel phenomena between hemin-H(2)O(2)-NO(2)(-) and 3-morpholinosydnonimine hydrochloride (SIN-1)-mediated oxidation and nitration reactions of glutamate dehydrogenase (GDH). Hemin-H(2)O(2) could effectively induce GDH protein oxidation and reduce its activity. Although the addition of low concentration of nitrite promoted protein oxidation, protein oxidation was weakened with the increase of nitrite concentration, meanwhile, tyrosine nitration was increased and the enzyme activity was partially restored. However, with the increase of SIN-1 concentration, protein oxidation and tyrosine nitration were increased, enzyme activity was decreased. The presence of desferrioxamine and/or catechin inhibit tyrosine nitration both in hemin-H(2)O(2)-NO(2)(-) and in SIN-1, but they promoted protein oxidation and reduced the enzyme activity in hemin-H(2)O(2)-NO(2)(-) system, while inhibited protein oxidation and recover the enzyme activity in SIN-1 system. These results reveal both hemin-H(2)O(2)-NO(2)(-) and SIN-1 can cause inactivation of GDH through protein oxidation and tyrosine nitration, but the impact of the effect of protein oxidation (not thiol oxidation) on enzyme activity is stronger than that of protein tyrosine nitration. Moreover, mass spectrometric analysis indicated that nitrated tyrosine residues by hemin-H(2)O(2)-NO(2)(-) were Tyr262 and Tyr471 while by SIN-1 were Tyr401 and Tyr493. It meant that protein oxidation and tyrosine nitration of GDH induced by hemin-H(2)O(2)-NO(2)(-) were different from those induced by SIN-1.     

The effect of HOCl-induced modifications on phosphatase and tensin homologue (PTEN) structure and function

Oxidation by reactive species can cause changes in protein function and affect cell signalling pathways. Phosphatase and tensin homologue (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidised; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilisation and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.     

The effect of neighboring amino acid residues and solution environment on the oxidative stability of tyrosine in small peptides

The effects of neighboring residues and formulation variables on tyrosine oxidation were investigated in model dipeptides (glysyl tyrosine, N-acetyl tyrosine, glutamyl tyrosine, and tyrosyl arginine) and tripeptide (lysyl tyrosyl lysine). The tyrosyl peptides were oxidized by light under alkaline conditions by a zero-order reaction. The rate of the photoreaction was dependent on tyrosyl pK(a), which was perturbed by the presence of neighboring charged amino acid residues. The strength of light exposure, oxygen headspace, and the presence of cationic surfactant, cetyltrimethylammonia chloride had a significant effect on the kinetics of tyrosyl photo-oxidation. Tyrosine and model tyrosyl peptides were also oxidized by hydrogen peroxide/metal ions at neutral pH. Metal-catalyzed oxidation followed first-order kinetics. Adjacent negatively charged amino acids accelerated tyrosine oxidation owing to affinity of the negative charges to metal-ions, whereas positively charged amino acid residues disfavored the reaction. The oxidation of tyrosine in peptides was greatly affected by the presence of adjacent charged residues, and the extent of the effect depended on the solution environment.     

Periodate oxidation of sperm-whale myoglobin and the role of the methionine residues in the antigen–antibody reaction

1. Oxidation of sperm-whale metmyoglobin and its apoprotein with periodate has been investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH6.8 and 22 degrees consumption of periodate ceased in 3(1/2)hr. at 43 moles of periodate/mole of myoglobin. The two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues were oxidized; serine increased in the hydrolysates from 6 to 9 residues/mol. 3. At pH5.0 and 22 degrees , consumption levelled off in 4(1/2)hr. at 26 moles of periodate/mole of myoglobin and resulted in the modification of the two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues; serine increased from 6 to 7 residues/mol. and, also, ferrihaem suffered considerable oxidation. 4. Oxidation at pH5.0 and 0 degrees resulted at completion (4hr.) in the consumption of 22 moles of periodate/mole of myoglobin and in the modification of the methionine, tyrosine and tryptophan residues. Spectral studies indicated oxidation of the haem group. This derivative reacted very poorly with rabbit antisera to MbX (the major component no. 10 obtained by CM-cellulose chromatography; Atassi, 1964). 5. Oxidation of apomyoglobin at pH5.0 and 0 degrees was complete in 4hr. with the consumption of 7.23 moles of periodate/mole of apoprotein. The rate of oxidation in decreasing order was: methionine; tryptophan; tyrosine; and after 7hr. of reaction the following residues/mol. were oxidized: methionine, 2.0; tryptophan, 1.6; tyrosine, 0.99. No peptide bonds were cleaved. Metmyoglobin prepared from the 7hr.-oxidized apoprotein showed that the reactivity with antisera to MbX had diminished considerably. 6. Milder oxidation of apoprotein (2 molar excess of periodate, pH5.0, 0 degrees , 2hr.) resulted in the modification of 1.66 residues of methionine/mol. Metmyoglobin prepared from this apoprotein was identical with native MbX spectrally, electrophoretically and immunochemically. It was concluded that the methionine residues at positions 55 and 131 were not essential parts of the antigenic sites of metmyoglobin.     

Raman and infrared spectra of toxin gamma from the venom of the scorpion Tityus serrulatus

Toxin gamma is a basic, low-molecular-weight, neurotoxic protein, isolated from the venom of the Brazilian scorpion, Tityus serrulatus. Raman spectra (400-1800 cm-1 region) of this toxin in both the lyophilized state and in 0.1 M acetate buffer (pH 4.5) and the infrared spectrum (700-4000 cm-1 region) of a solid film were investigated. From the vibrational spectra, it can be concluded that the polypeptide backbone of toxin gamma consists of a mixture of the different secondary structures, with predominance of beta-sheet, followed by unordered structure and alpha-helix, with some evidence of beta-turn structures. The four disulfide bridges assume the gauche-gauche-gauche conformation of the CCSSCC fragments. The intensity ratio of the doublet at 853 and 828 cm-1 suggests that four out of the five tyrosine residues are exposed. The three tryptophan residues are exposed on the surface, and the single methionine residue assume the gauche-gauche conformation. Toxin gamma retains full activity in the pH 4.5-7.5 range, but is almost completely inactivated at pH 11.5.     

Reversible oxidation of the membrane distal domain of receptor PTPalpha is mediated by a cyclic sulfenamide

Protein tyrosine phosphatases (PTPs) are fundamental to the regulation of cellular signalling cascades triggered by protein tyrosine kinases. Most receptor-like PTPs (RPTPs) comprise two tandem PTP domains, with only the membrane proximal domains (D1) having significant phosphatase activity; the membrane distal domains (D2) display little to no catalytic activity. Intriguingly, however, many RPTP D2s share the catalytically essential Cys and Arg residues of D1s. D2 of RPTPalpha may function as a redox sensor that mediates regulation of D1 via reactive oxygen species. Oxidation of Cys723 of RPTPalpha D2 (equivalent to PTP catalytic Cys residues) stabilizes RPTPalpha dimers, induces rotational coupling, and is required for inactivation of D1 phosphatase activity. Here, we investigated the structural consequences of RPTPalpha D2 oxidation. Exposure of RPTPalpha D2 to oxidants promotes formation of a cyclic sulfenamide species, a reversibly oxidized state of Cys723, accompanied by conformational changes of the D2 catalytic site. The cyclic sulfenamide is highly resistant to terminal oxidation to sulfinic and sulfonic acids. Conformational changes associated with RPTPalpha D2 oxidation have implications for RPTPalpha quaternary structure and allosteric regulation of D1 phosphatase activity.     

Functional consequences of tryptophan modification in human fibrinogen.

When human fibrinogen was modified with H2O2, inter- and intra-molecular cross-links of fibrinogen were formed, accompanied with oxidation of tryptophan, methionine and tyrosine residues. These cross-links may be closely associated with oxidation of tryptophan residues. The polymerization activity of fibrinogen with thrombin was decreased markedly by this modification. Modification of tryptophan residues in fibrinogen was also performed with 2-hydroxy-5-nitrobenzyl bromide. Modification of two out of a total 78 tryptophan residues in the molecule with the reagent led to the intensification (1.7 times) of the polymerization activity with thrombin and further modification of the next two residues led to complete loss of the polymerization activity. The first two tryptophan residues to be modified are in Fragment D, and the next two occur in Fragment E.     

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.     

Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives

The 13 amino acid toxic peptide from the marine snail Conus geographus, conotoxin GI, blocks the acetylcholine receptor at the neuromuscular junction. In this report, we describe a method for analyzing disulfide bonding in nanomole amounts of small cystine-rich peptides. The procedure involves partial reduction and a double-label alkylation of cysteine residues. Using this method, we show that the natural conotoxin GI has a (2-7, 3-13) disulfide configuration. The structure of conotoxin GI has been confirmed by chemical synthesis. The preparation and purification of molecularly homogeneous, iodinated derivatives of this toxin are also described. All derivatives, including the [diiodohistidine,diiodotyrosine]conotoxin GI, retained at least half of the biological activity of unmodified toxin. Since the tetraiodinated toxin, which is greater than 25% by weight iodine, retains considerable toxicity, unmodified histidine and tyrosine residues in conotoxin GI are not crucial for biological activity.     

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFβR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFβR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFβR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFβR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.     

Voltammetry of tobacco mosaic virus and its isolated protein at the graphite electrode

The electrochemical behaviour of tobacco mosaic virus (TMV) and its isolated protein was studied using differential pulse (DP) voltammetry at a graphite electrode and by direct current (DC) polarography in Brdicka solution. TMV and its isolated protein were found to be electrooxidized at the graphite electrode in the adsorbed state. Both species yielded two oxidation peaks on DP voltammograms. The first, more negative peak, corresponded to electrooxidation of tyrosine residues, whereas the other, more positive, peak corresponded to electrooxidation of tryptophan residues. DC polarography was used to detect degradation of TMV and denaturation of TMV-protein induced by an increased pH and by the addition of urea, respectively. These structural transformations resulted in increased DP voltammetric oxidation currents as recorded using a graphite working electrode. It has been suggested that the higher oxidation currents were due to an increase in the number of tyrosine and tryptophan residues accessible to the reaction at the graphite electrode. The results of these electrochemical investigations were in a good agreement with the estimation of the accessibility of tyrosine and tryptophan residues based on the well-explored three-dimensional structure of TMV and its isolated protein.     

Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

We have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Tyr-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater than the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by [3H]diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.     

Studies on the toxin of Aspergillus fumigatus. XVIII. Photooxidation of asp-hemolysin in the presence of various dyes and its relation to the site of hemolytic activity

The site of hemolytic activity of a toxin isolated from Aspergillus fumigatus designated Asp-hemolysin was determined by photooxidation techniques. The hemolytic activity of this toxin was strongly inhibited by photooxidation with methylene blue, rose bengal, riboflavin, or eosin G as a sensitizer, whereas crystal violet, hematoxylin, naphthol yellow S, bromothymol blue, methyl orange, and cresol red had no effect. pH dependence of the inactivation with methylene blue was observed in the narrow range of pH values from 7.0 to 8.0, like that of the inactivation with rose bengal or riboflavin. The histidine, cysteine, methionine, tryptophan, and tyrosine content of methylene blue-photooxidized Asp-hemolysin was significantly decreased, while other amino acids were not affected. The hemolytic activity of the toxin was lost more slowly than the histidine residue, being maintained at about 50% even at the time when the histidine residue was completely lost after 30 min. Photooxidation of Asp-hemolysin in the presence of rose bengal also caused a decrease in histidine, methionine, and threonine content. These findings suggest that residues of cysteine, methionine, threonine, tryptophan, and/or tyrosine but not histidine may play an important role through stereostructure in the manifestation of the hemolytic activity of Asp-hemolysin.     

Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.     

Studies on tryptophan residues of Abrus agglutinin. Stopped-flow kinetics of modification and fluorescence-quenching studies.

The presence of two essential tryptophan residues/molecule was implicated in the binding site of Abrus agglutinin [Patanjali, Swamy, Anantharam, Khan & Surolia (1984) Biochem. J. 217, 773-781]. A detailed study of the stopped-flow kinetics of the oxidation of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues into two phases was observed upon ligand binding. The heterogeneity of tryptophan exposure was substantiated by quenching studies with acrylamide, succinimide and Cs+. Our study revealed the microenvironment of tryptophan residues to be hydrophobic, and also the presence of acidic amino acid residues in the vicinity of surface-localized tryptophan residues.     

The influence of chemical modification on the biological properties of cloacin DF13

The influence of chemical modification on the biological properties of the bacteriocin cloacin DF13 has been investigated. All chemical modifications resulted in the total loss of the ability of the bacteriocin to kill sensitive bacterial cells. The ability of the bacteriocin to bind to specific receptor sites on sensitive bacteria was affected by the modification of carboxyl groups with glycine ethyl ester (GEE) and by the oxidation of tryptophan residues with N-bromosuccinimide (NBS). The endoribonucleolytic activity of the bacteriocin was affected by nitration of tyrosine residues with tetranitromethane (TNM) or by the oxidation of tryptophan residues with NBS. Binding of immunity protein to the cloacin was not affected by either of these modifications.     

Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site

4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.