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Journal of the American Chemical Society Mar 2023Ribonucleotide reductase (RNR) regulates DNA synthesis and repair in all organisms. The mechanism of RNR requires radical transfer over a proton-coupled electron...
Ribonucleotide reductase (RNR) regulates DNA synthesis and repair in all organisms. The mechanism of RNR requires radical transfer over a proton-coupled electron transfer (PCET) pathway spanning ∼32 Å across two protein subunits. A key step along this pathway is the interfacial PCET reaction between Y356 in the β subunit and Y731 in the α subunit. Herein, this PCET reaction between two tyrosines across an aqueous interface is explored with classical molecular dynamics and quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The simulations suggest that the water-mediated mechanism involving double proton transfer through an intervening water molecule is thermodynamically and kinetically unfavorable. The direct PCET mechanism between Y356 and Y731 becomes feasible when Y731 is flipped toward the interface and is predicted to be approximately isoergic with a relatively low free energy barrier. This direct mechanism is facilitated by the hydrogen bonding of water to both Y356 and Y731. These simulations provide fundamental insights into radical transfer across aqueous interfaces.
Topics: Tyrosine; Protons; Ribonucleotide Reductases; Electrons; Models, Molecular; Electron Transport; Escherichia coli; Water
PubMed: 36802630
DOI: 10.1021/jacs.2c13615 -
Methods in Enzymology 2008Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells... (Review)
Review
Protein 3-nitrotyrosine in complex biological samples: quantification by high-pressure liquid chromatography/electrochemical detection and emergence of proteomic approaches for unbiased identification of modification sites.
Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues.
Topics: Animals; Chromatography, High Pressure Liquid; Complex Mixtures; Electrochemistry; Humans; Proteins; Proteomics; Tandem Mass Spectrometry; Tyrosine
PubMed: 18554526
DOI: 10.1016/S0076-6879(08)01201-9 -
The Journal of Biological Chemistry Oct 2001EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon...
EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.
Topics: Amino Acid Sequence; Binding Sites; Cell Movement; Cells, Cultured; Cytoplasm; Ephrin-B1; Ephrin-B2; Humans; Immunoblotting; Ligands; Mass Spectrometry; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Precipitin Tests; Protein Binding; Retina; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Transfection; Tyrosine
PubMed: 11466320
DOI: 10.1074/jbc.M105815200 -
The Biochemical Journal Feb 2000Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of...
Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of nitrotyrosine in vivo has been reported with the use of a variety of methods including immunoassay, HPLC and GLC/MS. The validity of HPLC and immunoassays have been questioned with regard to their selectivity and sensitivity limits. In principle, the measurement of nitrotyrosine by GLC/MS permits a highly specific, highly sensitive and fully quantitative assay. The nitration of tyrosine under acidic conditions in the presence of nitrite is well documented. Derivatization for the full quantification of nitrotyrosine by using GLC/MS can lead to the artifactual nitration of tyrosine if performed under acidic conditions in the presence of nitrite. We describe a novel alkaline method for the hydrolysis and derivatization of nitrotyrosine and tyrosine, and demonstrate its applicability to the measurement of plasma concentrations of both free and protein-bound nitrotyrosine and tyrosine. A detection limit of 1 pg for nitrotyrosine and 100 pg for tyrosine has been achieved. Our method allows, for the first time, the analysis of free and protein-bound nitrotyrosine and tyrosine in biological samples. The plasma concentrations (means+/-S.E.M.) of free tyrosine and nitrotyrosine in eight normal subjects were 12+/-0.6 microg/ml and 14+/-0.7 ng/ml respectively. Plasma proteins contained tyrosine and nitrotyrosine at 60.7+/-1.7 microg/mg and 2.7+/-0.4 ng/mg respectively.
Topics: Adult; Artifacts; Blood Proteins; Gas Chromatography-Mass Spectrometry; Humans; Male; Nitrates; Tyrosine
PubMed: 10642501
DOI: No ID Found -
Biochemical and Biophysical Research... Nov 2009Previously we identified threonine-1172 (T1172) in the cytoplasmic domain of the cell adhesion molecule L1 as phosphorylated in pancreatic cancer cells. Although both...
Previously we identified threonine-1172 (T1172) in the cytoplasmic domain of the cell adhesion molecule L1 as phosphorylated in pancreatic cancer cells. Although both CKII- and PKC-blockade suppressed this modification, only CKII was capable of phosphorylating T1172 of a recombinant L1 cytoplasmic domain, suggesting the requirement for additional events to facilitate availability of T1172 to PKC. In this study, we demonstrate that the region around T1172 exists in distinct conformations based on both T1172 phosphorylation and the integrity of surrounding residues. We further demonstrate the role of membrane-proximal and membrane-distal residues in regulating cytoplasmic domain conformation, and that modification of 3 of the 4 tyrosines in the L1 cytoplasmic domain promote conformational changes that facilitate other events. In particular, phenylalanine-substitution of tyrosine-1151 or tyrosine-1229 promote opening up of the cytoplasmic domain in a manner that facilitates phosphorylation of the other 3 tyrosines, as well as phosphorylation of T1172 by PKCalpha. Importantly, we show that phosphorylation of serine-1181 is required for T1172 phosphorylation by CKII. These data define a specific role for secondary structure in regulating the availability of T1172 that facilitates phosphorylation by PKC.
Topics: Amino Acid Sequence; Antibodies, Phospho-Specific; Casein Kinase II; Cytoplasm; Epitopes; Humans; Molecular Sequence Data; Mutation; Neural Cell Adhesion Molecule L1; Phosphorylation; Protein Folding; Protein Kinase C-alpha; Protein Structure, Secondary; Protein Structure, Tertiary; Serine; Threonine; Tyrosine
PubMed: 19720049
DOI: 10.1016/j.bbrc.2009.08.143 -
Blood Sep 1994Fc gamma RIIA in the absence of other Fc receptors or receptor subunits induces the ingestion of IgG-coated cells. The cytoplasmic domain of Fc gamma RIIA contains two...
Fc gamma RIIA in the absence of other Fc receptors or receptor subunits induces the ingestion of IgG-coated cells. The cytoplasmic domain of Fc gamma RIIA contains two Y-x-x-L sequences similar to those in other Ig gene family receptors plus an additional tyrosine residue not in a Y-x-x-L motif. Upon cross-linking, Fc gamma RIIA is phosphorylated on tyrosine and the cytoplasmic tyrosines, Y275 (Y1), Y282 (Y2), and Y298 (Y3), may be important for its phagocytic activity. Because COS-1 cells can serve as a model for examining molecular structures involved in phagocytosis, substitutions and deletions were introduced into the cytoplasmic domain of Fc gamma RIIA and examined in COS-1 cell transfectants for their effects on phagocytosis and tyrosine phosphorylation. Disruption of a single cytoplasmic Y-x-x-L motif by substitution of tyrosine Y2 or Y3 by phenylalanine or by removing the threonine and leucine residues within the motif inhibited phagocytosis 50% to 65%. Tyrosine phosphorylation of Fc gamma RIIA also was inhibited, although to a greater extent by the substitution of Y3 than of Y2. Replacement of the N-terminal first cytoplasmic domain tyrosine, Y1, which is not within a typical Y-x-x-L, by itself did not inhibit phagocytosis, but replacement of Y1 in mutants lacking Y2 or Y3 virtually eliminated phagocytic activity and receptor tyrosine phosphorylation. Thus, at least two cytoplasmic tyrosines, including at least one typical single Y-x-x-L motif, are required for phagocytosis by Fc gamma RIIA. The data suggest that there is a close but not a simple relationship between phosphorylation of the Fc gamma RIIA cytoplasmic tyrosines and Fc gamma RIIA-mediated phagocytosis. Y3 appears to be particularly important because its removal by truncation or replacement with phenylalanine inhibits both tyrosine phosphorylation and phagocytosis in parallel. Alterations in the 12 residue proline-containing sequence between the two Y-x-x-L motifs also reduced phagocytic activity and tyrosine phosphorylation. Thus, the specific structure of the Fc gamma RIIA cytoplasmic domain accounts for its ability to stimulate phagocytosis in the absence of other subunits.
Topics: Amino Acid Sequence; Animals; Antigens, CD; Cell Line; Cytoplasm; Humans; Molecular Sequence Data; Mutagenesis; Phagocytosis; Phenylalanine; Phosphorylation; Phosphotyrosine; Receptors, IgG; Structure-Activity Relationship; Transfection; Tyrosine
PubMed: 7521687
DOI: No ID Found -
The Journal of Biological Chemistry Apr 2002Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of...
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.
Topics: Catalysis; Dopamine; Electrophoresis, Polyacrylamide Gel; Humans; Mutagenesis, Site-Directed; Mutation; Nitrogen; Peroxynitrous Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tyrosine; Tyrosine 3-Monooxygenase
PubMed: 11834745
DOI: 10.1074/jbc.M200290200 -
The Journal of Clinical Investigation Jun 1993Phagocytes generate H2O2 for use by a secreted heme enzyme, myeloperoxidase, to kill invading bacteria, viruses, and fungi. We have explored the possibility that...
Phagocytes generate H2O2 for use by a secreted heme enzyme, myeloperoxidase, to kill invading bacteria, viruses, and fungi. We have explored the possibility that myeloperoxidase might also convert L-tyrosine to a radical catalyst that cross-links proteins. Protein-bound tyrosyl residues exposed to myeloperoxidase, H2O2, and L-tyrosine were oxidized to o,o'-dityrosine, a stable product of the tyrosyl radical. The cross-linking reaction required L-tyrosine but was independent of halide and free transition metal ions; the heme poisons azide and aminotriazole were inhibitory. Activated neutrophils likewise converted polypeptide tyrosines to dityrosine. The pathway for oxidation of peptide tyrosyl residues was dependent upon L-tyrosine and was inhibited by heme poisons and catalase. Dityrosine synthesis was little affected by plasma concentrations of Cl- and amino acids, suggesting that the reaction pathway might be physiologically relevant. The requirement for free L-tyrosine and H2O2 for dityrosine formation and the inhibition by heme poisons support the hypothesis that myeloperoxidase catalyzes the cross-linking of proteins by a peroxidative mechanism involving tyrosyl radical. In striking contrast to the pathways generally used to study protein oxidation in vitro, the reaction does not require free metal ions. We speculate that protein dityrosine cross-linking by myeloperoxidase may play a role in bacterial killing or injuring normal tissue. The intense fluorescence and stability of biphenolic compounds may allow dityrosine to act as a marker for proteins oxidatively damaged by myeloperoxidase in phagocyte-rich inflammatory lesions.
Topics: Amino Acid Sequence; Cross-Linking Reagents; Dipeptides; Free Radicals; Humans; Molecular Sequence Data; Neutrophils; Oligopeptides; Oxidation-Reduction; Peroxidase; Serum Albumin, Bovine; Tyrosine
PubMed: 8390491
DOI: 10.1172/JCI116531 -
Journal of Photochemistry and... 2011In this article, progress in understanding proton coupled electron transfer (PCET) in Photosystem II is reviewed. Changes in acidity/basicity may accompany... (Review)
Review
In this article, progress in understanding proton coupled electron transfer (PCET) in Photosystem II is reviewed. Changes in acidity/basicity may accompany oxidation/reduction reactions in biological catalysis. Alterations in the proton transfer pathway can then be used to alter the rates of the electron transfer reactions. Studies of the bioenergetic complexes have played a central role in advancing our understanding of PCET. Because oxidation of the tyrosine results in deprotonation of the phenolic oxygen, redox active tyrosines are involved in PCET reactions in several enzymes. This review focuses on PCET involving the redox active tyrosines in Photosystem II. Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone. Photosystem II provides a paradigm for the study of redox active tyrosines, because this photosynthetic reaction center contains two tyrosines with different roles in catalysis. The tyrosines, YZ and YD, exhibit differences in kinetics and midpoint potentials, and these differences may be due to noncovalent interactions with the protein environment. Here, studies of YD and YZ and relevant model compounds are described.
Topics: Electron Transport; Kinetics; Oxidation-Reduction; Photosystem II Protein Complex; Plastoquinone; Protons; Tyrosine
PubMed: 21419640
DOI: 10.1016/j.jphotobiol.2011.01.026 -
European Journal of Biochemistry Mar 1997The interactions of ring fluorinated analogs of tyrosine with tyrosine phenol-lyase and tryptophan indole-lyase (tryptophanase) were studied by rapid-scanning...
The interactions of ring fluorinated analogs of tyrosine with tyrosine phenol-lyase and tryptophan indole-lyase (tryptophanase) were studied by rapid-scanning stopped-flow spectrophotometry. The reaction of L-tyrosine with tyrosine phenol-lyase resulted in rapid formation of a small absorbance peak at 500 nm, attributed to a quinonoid intermediate. The reaction of 3-fluoro-L-tyrosine with tyrosine phenol-lyase resulted in a peak at 500 nm with much higher absorbance, as did the reaction of 3,5-difluoro-L-tyrosine, due to increased accumulation of quinonoid intermediates. In constrast, complexes with 2-fluoro-L-tyrosine, 2,3-difluoro-L-tyrosine, 2,5-difluoro-L-tyrosine, and 2,6-difluoro-L-tyrosine exhibited much lower absorbance intensity at 500 nm. The rate constant for quinonoid intermediate formation from 3-fluoro-L-tyrosine was comparable to that for L-tyrosine. However, 3,5-difluoro-L-tyrosine reacted to form a quinonoid intermediate at about half the rate of L-tyrosine, while 2,3-difluoro-L-tyrosine reacted at twice the rate of L-tyrosine. In addition, the 2-substituted difluorotyrosines exhibited an intermediate, which was formed rapidly, absorbing strongly at about 340 nm, which is likely due to a gem-diamine intermediate. Tyrosine is not a substrate for tryptophan indole-lyase; the reaction of tryptophan indole-lyase with L-tyrosine resulted in formation of external aldimine, which absorbed at 420 nm, and a very small absorbance peak at 500 nm. 3-Fluoro-L-tyrosine reacted with tryptophan indole-lyase to produce a prominent quinonoid absorbance peak at 500 nm, whereas L-tyrosine, 2-fluoro-L-tyrosine, and all difluoro-L-tyrosines, had a much reduced intensity for this peak. Thus, the presence of ring fluorine substituents in L-tyrosine that are remote from the site of the chemical transformation has significant effects on the rates and equilibria of intermediate formation in the reactions with both tyrosine phenol-lyase and tryptophan indole-lyase. Although it is commonly thought that fluorine substitution will not result in any significant steric effects, our results suggest that the effects of fluorine substitution in the reactions of fluorinated tyrosines with tyrosine phenol-lyase and tryptophan indole-lyase are due to a combination of steric and electronic effects.
Topics: Citrobacter freundii; Escherichia coli; Fluorine; Kinetics; Molecular Structure; Spectrophotometry; Tryptophanase; Tyrosine; Tyrosine Phenol-Lyase
PubMed: 9119037
DOI: 10.1111/j.1432-1033.1997.00658.x