Chem. Res. Toxicol. 2004, 17, 1323-1328
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Thiyl Radical Reaction with Amino Acid Side Chains: Rate Constants for Hydrogen Transfer and Relevance for Posttranslational Protein Modification Thomas Nauser,†,‡ Jill Pelling,§ and Christian Scho¨neich*,† Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047, and Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160-7410 Received May 24, 2004
Thiyl radicals are prominent intermediates during biological conditions of oxidative stress and have been suggested to be involved in the mutagenic effects of thiols. While several enzymatic processes rely on the formation and selective reactions of protein thiyl radicals with substrates, such reactions may represent a source for biological damage when occurring uncontrolled during oxidative stress. For example, intramolecular hydrogen transfer reactions to protein cysteine thiyl radicals may lead to secondary amino acid oxidation products, which may represent starting points for protein aggregation and/or fragmentation. Here, we have used a kinetic NMR method to determine rate constants, ksc, for hydrogen transfer reactions between thiyl radicals and amino acid side chain C-H bonds at 37 °C. Rate constants cover a range between ksc e 1 × 103 M-1 s-1 (Val) and ksc ) 1.6 × 105 M-1 s-1 (Ser). On the basis of these values and earlier data, model calculations are performed, which will demonstrate that protein thiyl radicals may attack protein C-H bonds via intramolecular hydrogen transfer at physiological conditions, potentially resulting in irreversible protein damage.
Introduction (RS•
Thiyl radicals ) are important intermediates during biological conditions of oxidative stress (1). They are moderately good oxidants and have been suggested to mediate DNA degradation during the anaerobic reaction of glutathione with Cu(II) (2). Much less studied is the potential damaging effect of thiyl radicals on proteins. Such reactions may be of greater biological significance as they can occur intramolecularly. In general, Cys residues represent facile targets for oxidation by various reactive oxygen species. The tumor suppressor protein p53 contains highly reactive Cys residues (3), but the oxidation with peroxynitrite yields nonreducible aggregates (which are not dityrosine) (4) suggesting a covalent aggregation mechanism, which does not involve disulfide bonds. In this respect, the hydrogen transfer reaction between a Cys thiyl radical and a neighboring amino acid may become important as a starting point for alternative covalent aggregation mechanisms involving secondary radicals and products (e.g., carbonyls). In the sequence of human p53 (NCBI NP 000537), seven out of ten Cys residues are in the vicinal position (i.e., in position i ( 1) to Thr, Ser, Phe, or Met residues, which contain side chains with activated C-H bonds. In addition, four Cys residues are located in position i ( 2, three in position i ( 3, and one in position i ( 4 relative to Thr, Ser, Phe, and Met. Thus, several possibilities exist for Cys residues to interact with these amino acids. The * To whom correspondence should be addressed. Tel: 785-864-4880. Fax: 785-864-5736. E-mail:
[email protected]. † University of Kansas. ‡ Present address: Laboratory for Inorganic Chemistry, ETH Zu ¨ rich, CH-8093 Zu¨rich, Switzerland. § University of Kansas Medical Center.
probabilities for such interactions depend on the threedimensional structure, flexibilities of the respective protein domains, and steric effects. Each of these amino acids may suffer hydrogen abstraction such as displayed in equilibrium 1, where X represents an aryl, alkylthio, alkoxy, or hydroxyl group.
Radiation chemical and kinetic NMR studies have yielded absolute rate constants k1 ) 3 × 103 to 3 × 104 M-1 s-1 for the hydrogen abstraction by thiyl radicals from several aliphatic alcohols, ethers, and carbohydrates (5-7). These rate constants are small as compared to those for the reverse reaction, k-1 ) 107-108 M-1 s-1 (8). However, rate constants for amino acid side chains are not available and are, therefore, the focus of this paper. To quantitatively predict potential hydrogen transfer reactions between thiyl radicals and amino acid side chains in proteins, we have used a kinetic NMR method to measure rate constants between thiyl radicals and various amino acid substrates. We have selected free amino acids and amino acid amides at acidic pH values. Protonation of a free amino acid renders the RC-H bonds fairly unreactive toward hydrogen transfer (9); that is, these reaction conditions permit the quite selective measurement of hydrogen transfer reactions of the amino acid side chains. For practical reasons, we included the measurements of amino acid amides, which cannot adopt zwitterionic structures, so that small variations of the experimental pD between 3.0 and 3.5 did not change any ratios of cationic to zwitterionic substrate.
10.1021/tx049856y CCC: $27.50 © 2004 American Chemical Society Published on Web 08/14/2004
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Chem. Res. Toxicol., Vol. 17, No. 10, 2004
Scheme 1. Production and Reactions of Cysteamine Thiyl Radicals (2-Aminoethylthiyl Radicals, CyaS•) with Amino Acids (AAH) and a Reference Compound (RefH)a
Nauser et al. Table 1. Rate Constants for Overall Hydrogen Transfer, k4, and Hydrogen Transfer from the Side Chain, ksc, of Amino Acids and Amino Acid Amides by Thiyl Radicals at 37 °C and pD 3-3.4
AAH Ala Gly His Met Phe Ser Ser
a
All experiments were carried out in oxygen free D2O solutions.
We will demonstrate that especially Ser and Thr show unexpectedly high rate constants on the order of 105 M-1 s-1, suggesting that these amino acids will be highly susceptible targets for thiyl radicals in proteins. The reactivities of these side chains will be compared to the reactivities of the RC-H bonds in the amino acid substrates. Furthermore, in kinetic models, we will demonstrate that these hydrogen transfer reactions are relevant for conditions of oxidative stress in vivo.
Experimental Section Materials. All chemicals were purchased in the highest commercially available qualities and used as received with the exception of 2-propanol, which was redistilled. We obtained deuterium oxide (D2O) from Cambridge Isotope Laboratories (Andover, MA); 2-propanol was obtained from Fisher (Chicago, IL); 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), cysteamine hydrochloride (2-aminoethanetiol, CyaSH), sulfuric acid, and sarcosine anhydride were obtained from Sigma-Aldrich (St. Louis, MO); and all amino acids, their amides, and NAcGlyNH2 were obtained from Bachem (King of Prussia, PA). Reaction Conditions. The kinetic NMR method has been described and validated in detail earlier (7, 10, 11). Briefly, AAPH was incubated at 37 °C in a deaerated solution of D2O containing H/D-exchanged cysteamine (0.5-5 mM CyaSD), a reference competitor, and the amino acid substrate. Scheme 1 summarizes all key reactions. The AAPH-derived carboncentered radicals abstract deuterium from CyaSD to yield thiyl radicals, CyaS•, which, in turn, abstract hydrogen atoms from the reference competitor (reaction 3; k3 is known) or from the amino acid (reaction 4). In the reverse reactions, deuterium is incorporated into the molecules, resulting in a decrease of the respective 1H NMR response. All 1H NMR spectra were measured on a Bruker Advance 400 MHz instrument. As competitors, we used 2-40 mM 2-propanol, 1-20 mM N-AcGlyNH2, or sarcosine anhydride. Amino acids and their corresponding amides were used at concentrations of 1-100 mM. Our acidic reaction conditions (pD 3.4) suppress the formation of (CyaSSCya)•- via reaction 5. Note that at this pD already more that 10% of the carboxylic acid group in the amino acids may be protonated. Calculated amounts of sulfuric acid were added to the solutions to adjust the desired pD, which was generally measured after the NMR measurements where pD ) pH + 0.4 (12).
Results Overall Rate Constants k4 for Hydrogen Abstraction from Amino Acid Substrates. The overall rate
Thr Thr Val AlaNH2 GlyNH2 HisNH2 MetNH2 PheNH2 SerNH2 SerNH2 ThrNH2 ValNH2
competitor
k4 × 104 (M-1 s-1)a (eq I)
k4 × 104 (M-1 s-1)a (eq II)
2-propanol 0.6 ( 0.2 2-propanol 0.3 ( 0.2 2-propanol 0.22 ( 0.05 2-propanol 1.7 ( 0.3 2-propanol 1.4 ( 0.2 AcGlyNH2 15 ( 10 30 ( 20 sarcosine 19 ( 10 anhydride AcGlyNH2 7.7 ( 1.5 2-propanol 2-propanol 0.4 ( 0.1 2-propanol 0.4 ( 0.1 2-propanol 0.7 ( 0.4 2-propanol 0.4 ( 0.1 2-propanol 1.9 ( 0.5 1.6 ( 0.8 2-propanol 1.5 ( 0.2 AcGlyNH2 25 ( 7 ThrNH2 20 ( 5 2-propanol 11 ( 5 8.9 ( 0.5 2-propanol 0.8 ( 0.3
kscb × 104 (M-1 s-1) ND ND 0.8 ( 0.5c (γC and C) g1.2d (βC) 12 ( 9e (βC) 14 ( 11f (βC) 4.6 ( 0.6g (βC)
