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PubMed1989

Tyrosine residues are essential for the activity of the human placental taurine transporter.

Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane greater than NBD-Cl greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. The dependence of taurine uptake rate on extravesicular Na+ concentration was sigmoidal and analysis of the data revealed that two Na+ ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na+-binding site of the transporter.

4-Chloro-7-nitrobenzofurazan↗
PubMed1978

Implication of a tyrosyl residue at the active site of mitochondrial L-malate:NAD+ oxidoreductase.

The tyrosyl residues of porcine mitochondrial L-malate:NAD+ oxidoreductase (EC 1.1.1.37) have been studied spectrophotometrically and using selective chemical modification with iodine and tetranitromethane. CNBr hydrolysis and Sephadex G-25, G-50 and G-75 chromatography produced a peptide which contained two tyrosines in the native and the nitrated molecules when the nitration took place in an NAD+-oxaloacetate solution. Nitration in the absence of the substrates caused the tyrosyl residues to disappear. Spectrophotometric titrations indicate that one of the 10 tyrosyl residues in mitochondrial L-malate:NAD+ oxidoreductase titrate abnormally, while iodination experiments suggest that two fast-reacting tyrosines are not involved in activity. Nitration and iodination experiments, in conjunction with CNBr-mapping, suggest that two of the four nitrated tyrosyl residues are necessary for biological action. Titration of the sulfhydryl groups with 4,4-bis-dimethylaminodiphenyl carbinol before and after nitration indicate that none of the cysteinyl residues were oxidized by the tetranitromethane, thus ruling out the loss of enzyme activity due to the thiol oxidation.

Amino Acids↗
PubMed1980

Involvement of tyrosyl residues in the substrate binding of pigeon liver malic enzyme.

The reactions of pigeon liver malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with tetranitromethane and N-acetylimidazole have been investigated to obtain information about the functional role of tyrosine residues in this enzyme. Incubation of the sulfhydryl-masked enzyme with tetranitromethane or N-acetylimidazole caused a time-dependent loss of all enzymatic activities of this enzyme. The absorption spectra of both the nitrated and acetylated enzyme indicated modification of tyrosine residues. The enzymatic activity of the acetylated enzyme was reversed by hydroxylamine. No amino group modification was observed. Preincubation of the enzyme with dicarboxylate substrate (or inhibitor), nucleotide coenzyme and divalent metal ions protected the enzyme against these reagents. The acetylated enzyme showed different kinetic properties from the native enzyme. The apparent Michaelis constants for malate and oxaloacetate increase by 2-5-fold. The binding between acetylated enzyme and NADPH was not abolished. These results strongly suggest the involvement of tyrosine residues in the dicarboxylic acid binding of malic enzyme.

Acetylation↗
PubMed1971

Nitration of the tyrosine in histone F1 in salt solutions and in F1-polyanion complexes.

The reaction of histone F1 with tetranitromethane was used to study the environment of the single tyrosine residue in the molecule. It was found that at a 10-fold molar excess of tetranitromethane over tyrosine approx. 40 min were needed to convert 85% of the tyrosine residues to nitrotyrosine. The rate and extent of nitration of F1 in dilute salt solutions was used as a reference against which the rate and extent of nitration under conditions known to affect the conformation of F1 was measured. In 1.0 M NaCl solutions the rate of nitration decreased, suggesting that the presence of salt may cause a conformational change in the tyrosine containing region of the histone. When the histone is complexed with polyanions such as DNA, RNA or poly-L-glutamic acid the accessibility of the tyrosine to nitration is markedly reduced. The results indicate that upon association with polyanions the non-cationic, tyrosine-containing, region in F1 undergoes a conformational change or alternatively, this tyrosine containing region is tightly associated with the polyanion.

Animals↗
PubMed1977

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.

Bacterial Proteins↗
PubMed1979

A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin.

The interaction of L-tryptophan and four benzodiazepine derivatives with tyrosine-modified human serum albumin was investigated by equilibrium dialysis and circular dichroism measurements. Out of the 18 tyrosine residues of the human serum albumin molecule, only 9 could be modified with tetranitromethane. At least up to a degree of modification of 5, the conformation of human serum albumin was not changed and no crosslinking and fractionation has been found, as revealed from circular dichroism measurements in the far ultraviolet range and from SDS polyacrylamide electrophoresis. The modification of only 2 out of the 9 accessible tyrosine residues of human serum albumin strongly affects the binding of L-tryptophan and diazepam to their common, stereospecific bindining site. This was evidently shown by a reduction of the association constants by more than 90% and by a large reduction of the extrinsic Cotton effects of four benzodiazepines bound to human serum albumin. The numbers of binding sites remained unchanged. Strong evidence was presented that only one tyrosine residue, which reacts faster with tetranitromethane than all others, is mainly involved in the specific indole and benzodiazepine binding site of human serum albumin. The location of this highly reactive tyrosine residue and that of the specific indole and benzodiazepine binding site within the human serum albumin primary structure is discussed.

Benzodiazepines↗
PubMed1980

The preparation of 3-nitrotyrosyl derivatives of three elapid venom cardiotoxins.

Nitration studies using tetranitromethane were conducted on the tyrosine residues of cardiotoxins, naja melanoleuca VII1, Naja haje annulifera VII1 and Hemachatus haemachates toxin 12B. Various partially and fully nitrated derivatives were formed. Analysis of the products of nitrating naja melanoleuca VII1 showed that the average relative reactivities of the three tyrosine residues were Tyr-25 greater than Tyr-22 greater than Tyr-51. It was significant that Tyr-51 could be easily modified in both N. melanoleuca VII1 and N. haje annulifera VII1. In contrast, other workers had found Tyr-51 in N. naja atra cardiotoxin to be unreactive towards tetranitromethane except under denaturing conditions. Fully nitrated derivatives of N. melanoleuca VII1 (Tyr-22, -25 and -51), N haje annulifera VII1 (Tyr-22 and -51) and H. haemachates 12B (Tyr-22), prepared under mild reaction conditions, were isolated by ion-exchange and hydrophobic interaction chromatographies. All three derivatives were pure by disc gel electrophoresis at pH 8.9 and amino acid analysis. They were therefore suitable for spectral and biological studies. The results were compared and contrasted to; those of other workers.

Amino Acids↗
PubMed1984

Essential tyrosyl residues of human placental alkaline phosphatase.

Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine. The inactivation process showed saturation kinetics. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate. The binding affinity between the modified enzyme and inorganic phosphate was decreased. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

Alkaline Phosphatase↗
PubMed1985

Partial chemical characterization of cyclopyrrolones ([3H] suriclone) and benzodiazepines ([3H]flunitrazepam) binding site: differences.

Rat hippocampus membranes were treated with several protein modifying reagents (iodoacetamide, N-ethylmaleimide, tetranitromethane and N-acetylimidazole). The effects of these treatments on the binding sites of cyclopyrrolones ([3H] suriclone), a new chemical family of minor tranquilizers, and benzodiazepines ([3H] flunitrazepam) were investigated. Here we show that both ligands are similarly sensitive to cysteine alkylation: [3H] suriclone and [3H] flunitrazepam binding are reduced by iodoacetamide and slightly increased by N-ethylmaleimide. On the contrary they are clearly differenciated by tyrosine modification: [3H] suriclone binding is not changed whereas [3H] flunitrazepam binding is increased by tetranitromethane and decreased by N-acetylimidazole. Our present findings and published evidence suggest cyclopyrrolones and benzodiazepines bind to distinct sites or to different allosteric forms of the benzodiazepine receptor.

Animals↗
PubMed1992

Modification of benzodiazepine receptors supports the distinctive role of histidine residues.

The effect of selective protein modifying reagents was examined on benzodiazepine (BZ) receptors in synaptosomal membrane preparations of rat whole brain and cerebellum. The potency of diethyl pyrocarbonate, a histidine modifying reagent, to inactivate BZ receptor binding, correlated with the rank order of agonist-inverse agonist efficacies of BZ ligands, the binding of the partial inverse agonist [3H]Ro 15-4513 was inactivated least. Diethyl pyrocarbonate slightly enhanced the displacing potency of Ro 15-4513 and enhanced its binding in low concentrations (1-2 mM). Diazepam-sensitive and -insensitive components of [3H]Ro 15-4513 binding were separated in cerebellum. Diethyl pyrocarbonate inactivated the diazepam-sensitive component with a potency (IC50 = 1.8 mM) similar to that on the binding of other benzodiazepines, while the diazepam-insensitive component was resistant to diethyl pyrocarbonate. Tetranitromethane and 2,3-butanedione (diacetyl), reagents specific for tyrosine and arginine residues respectively, exerted concentration-dependent partial inactivation of [3H]Ro 15-4513 binding. The diazepam-insensitive component of cerebellar Ro 15-4513 binding was more sensitive to inactivation by diacetyl but less sensitive to inactivation by tetranitromethane. These findings are consistent with a distinctive role of histidine-101 in alpha 1, alpha 2, alpha 3 and alpha 5 subunits of the gamma-aminobutyric acidA receptor complex and the His is replaced by an arginine residue in the alpha 6 subunit of the diazepam-insensitive cerebellar benzodiazepine receptors. The only other point of the protein sequence where histidine residues conserved in alpha 1, alpha 2, alpha 3 and alpha 5 subunits are replaced in alpha 6 is tyrosine-214 but this residue does not appear to contribute to benzodiazepine binding.

Animals↗
PubMed2004

Tyrosine residues modification studied by MALDI-TOF mass spectrometry.

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.

Amino Acid Substitution↗
PubMed1989

Amino acid residues essential for catalysis by peptidyl dipeptidase-4 from Pseudomonas maltophilia.

To assess residues essential for catalysis by prokaryotic peptidyl dipeptidase-4, the enzyme was subjected to chemical modification by a series of reagents. Treatment with either tetranitromethane or N-acetylimidazole abolished catalytic activity. Hydroxylamine reversed inactivation by acetylimidazole only. Thus, an essential tyrosine is indicated. Enzymatic activity also was quenched by either trinitrobenzenesulfonic acid or diethyl pyrocarbonate. Inactivation by these reagents was not reversed by hydroxylamine. These data suggest an essential lysine. The competitive inhibitor Phe-Arg protected partially against inactivation by tetranitromethane, and fully against inactivation by N-acetylimidazole. The substrate Hip-Phe-Arg protected against inactivation by trinitrobenzenesulfonic acid and diethyl pyrocarbonate. Thus, both tyrosine and lysine are located at the catalytic site.

Amino Acid Sequence↗
PubMed1987

F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane.

Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase. Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis. Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain. While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes. Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.

Amino Acid Sequence↗
PubMed1989

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity.

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.

Alcohol Oxidoreductases↗
PubMed1990

The ATP-binding site of the human placental H+ pump contains essential tyrosyl residues.

Transient exposure of human placental brush-border membrane vesicles to cholate reorients the ATP-driven H+ pump, enabling the pump to transport H+ into the vesicles upon addition of ATP to the external medium. H+ uptake can be measured in these vesicles by following the decrease in the absorbance of acridine orange, a delta pH indicator. We investigated the role of tyrosyl residues in the catalytic function of the H+ pump by studying the effects of tyrosyl group specific reagents on ATP-driven H+ uptake in cholate-pretreated membrane vesicles. The reagents tested were 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), N-acetylimidazole, tetranitromethane, and p-nitrobenzenesulfonyl fluoride. Treatment of the membrane vesicles with these reagents resulted in the inhibition of the ATP-driven H+ uptake, and the inhibitory potency was in the following order: NBD-Cl greater than tetranitromethane greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition of the H+ pump by NBD-Cl was reversible by 2-mercaptoethanol, and the inhibition by N-acetylimidazole was reversible by hydroxylamine. Since these reagents are not absolutely specific for tyrosyl groups and can also react with thiol groups, we studied the interaction of N-acetylimidazole with the H+ pump whose triol groups were masked by reaction with p-(chloromercuri)benzenesulfonate. The SH-masked pump was totally inactive, but the activity could be restored by dithiothreitol. On the contrary, the activity of the SH-masked H+ pump which was subsequently treated with N-acetylimidazole could not be restored by dithiothreitol, suggesting that thiol groups were not involved in the inhibition of the H+ pump by N-acetylimidazole.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗
PubMed1981

Proton magnetic resonance study of Streptomyces subtilisin inhibitor. pH titration and assignments of individual tyrosyl resonances.

This paper reports the pH titration at 25 degrees C and the assignment of aromatic proton resonances of three tyrosyl residues of each identical subunit of Streptomyces subtilisin inhibitor (Mr 23000) by high-resolution 1H NMR spectroscopy. The complete assignments of the specific tyrosyl resonances were made based on the results of the differential chemical modification of the tyrosyl residues with tetranitromethane followed by peptide analysis, independently of the knowledge of the crystal structure. pKa values of Tyr-7, -75, and -93 were determined in a 2H2O solution to be 10.95, 11.8, and greater than or equal to 12.6, respectively, at 25 degrees C, whereas pKa values of nitrated Tyr-7 and -75 were determined to be 7.3 and 7.9, respectively. Tyr-93 was not nitratable under normal conditions. The strong resistance to nitration, together with the extremely high pKa value and the high-field shifted positions of the ring proton resonances of Tyr-93 at neutral pH, strongly suggests that Tyr-93 takes part in a hydrogen bonding as a proton donor. Tyr-7 is more easily modified with tetranitromethane than Tyr-75, although in the crystal Tyr-75 is more exposed than Tyr-7. The result, together with the pKa value of Tyr-75 significantly higher than that of a normal tyrosine, indicates that the microenvironment of Tyr-75 is more restricted in solution than in solid. These results imply that structural details of a protein may be quite similar in solution to those in the crystal in the rigid hydrophobic region of the protein but that in the surface region of the protein local structures may well differ between the solution and the crystal.

Amino Acids↗
PubMed1975

Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney.

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.

Animals↗
PubMed1996

Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding.

The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.8M(-1.)min(-1) and 0.54M(-1.)min(-1) respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0M(-1.)min(-1). The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement or arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (deltaG')for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete activation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35mM compared with 10mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichromism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.

Arginine↗