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Protection of a Single-Cysteine Redox Switch from Oxidative Destruction: on the Functional Role of Sulfenyl Amide Formation in the Redox-Regulated Enzyme PTP1B

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Protection of a Single-Cysteine Redox Switch from Oxidative Destruction: on the Functional Role of Sulfenyl Amide Formation in the Redox-Regulated Enzyme PTP1B Bioorganic & Medicinal Chemistry Letters 20 (2010) 444–447 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Protection of a single-cysteine redox switch from oxidative destruction: On the functional role of sulfenyl amide formation in the redox-regulated enzyme PTP1B Santhosh Sivaramakrishnan, Andrea H. Cummings, Kent S. Gates * Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA article info abstract Article history: Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active Received 7 November 2009 site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this Revised 1 December 2009 enzyme against irreversible oxidative destruction. The results suggest that ‘overoxidation’ of the sulfenyl Accepted 2 December 2009 amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl Available online 4 December 2009 amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols. Ó 2009 Elsevier Ltd. All rights reserved. Keywords: Protein tyrosine phosphatase Redox regulation Sulfenic acid 10,11 Intracellular concentrations of hydrogen peroxide (H2O2) in- iii, Scheme 1B). Second, if ‘overoxidation’ does occur, the crease under conditions of oxidative stress and during some nor- resulting thiosulfinate likely could be converted cleanly back to mal signal transduction processes.1–3 An important mechanism the native cysteine residues by reactions with biological thiols by which cells issue temporary responses to such transitory in- (reaction v, Scheme 1B).14 creases in H2O2 levels involves reversible oxidation of cysteine res- Protein tyrosine phosphatases (PTPs) are important targets of 4,5 15–17 idues on critical ‘sensor’ proteins. The ability of cysteine residues intracellular H2O2. These cysteine-dependent enzymes cata- to serve as reversible redox switches relies upon the unique ability lyze the removal of phosphoryl groups from tyrosine residues on of the c–sulfur atom in this amino acid to cycle easily between (at their protein substrates.15–17 Accordingly, PTPs work in tandem least) two oxidation states under physiological conditions. Specifi- with protein tyrosine kinases to regulate critical signal transduc- cally, oxidation of a cysteine thiol by H2O2 yields a sulfenic acid tion cascades by modulating the phosphorylation status and, in residue (reaction i, Scheme 1A) that can, over time, be returned turn, the functional properties of proteins involved in these path- to the native thiol by reactions with biological thiols (reaction ii, ways.15–17 The catalytic activity of some PTPs is subject to redox Scheme 1A).4–8 Protein sulfenic acid residues also have the potential to undergo further reaction with hydrogen peroxide to generate the corre- native sulfenic sulfinic sponding sulfinic acid (reaction iii, Scheme 1A).4–9 This reaction A i iii cysteine H O acid acid is irreversible, except in the case of some peroxiredoxins8 and, 2 2 H2O2 therefore, yields an overoxidized, ‘broken’ redox switch. Alterna- 2RSH S S SH OH OOH tively, in some proteins, the initially-formed sulfenic acid interme- ii diate undergoes reaction with a neighboring ‘back door’ cysteine B i 10–13 thiol to generate a disulfide linkage (reaction ii, Scheme 1B). H2O2 Rudolph and Sohn provided evidence that, at least in the context SH SH SH S 2 OH of the phosphatase Cdc25B, disulfide formation protects the en- RS 4RSH v H ii zyme against irreversible overoxidation.10,11 There are at least iv two possible mechanisms underlying this protection. First, the S S disulfide may be relatively resistant to further oxidation (reaction H2O2 S S O iii thiosulfinate disulfide * Corresponding author. Tel.: +1 573 882 6763; fax: +1 573 882 2754. E-mail address: [email protected] (K.S. Gates). Scheme 1. 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.12.001 S. Sivaramakrishnan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 444–447 445 regulation as part of normal cell signaling processes.15–17 For O example, the enzyme PTP1B, a major negative regulator of the CO2Et O N O insulin signaling pathway, is inactivated by a burst of H2O2 that S CO2Et 18,19 CO2Et OO N is produced upon binding of insulin to its cell-surface receptor. N O or S S 2 Subsequent reactions with cellular thiols slowly return the enzyme O O to its active form.18,19 This transient oxidative inactivation of 1 3 PTP1B serves as a ‘timing device’ that increases phosphorylation levels on the insulin receptor and insulin receptor substrates, thus Scheme 3. potentiating cellular responses for a defined period of time follow- ing insulin stimulation.18,19 For some time, it was widely assumed that redox regulation of O O O PTPs involved either sulfenic acid or disulfide intermediates, as R R R'SH N R'SH N shown in Scheme 1. However, recent studies in the context of N R H H S PTP1B revealed a new mechanism for redox regulation of PTP S SR' SOH 2 O activity in which the initially-formed sulfenic acid undergoes reac- O R'SH tion with the neighboring amide nitrogen to yield a cyclic sulfenyl amide (known, more formally, as an isothiazolidin-3-one, reaction R = CH2CO2Et O O ii, Scheme 2).20–22 As required for a functional redox switch, reac- R R R' = CH2CH2OH N R'SH N tions with biological thiols can convert the sulfenyl amide back to H H SH S SR' the catalytically active thiol form of the enzyme (reaction iii, 54 Scheme 2).20–22 Importantly, sulfenyl amide formation subse- quently has been observed in other proteins and it has been sug- Scheme 4. gested that this posttranslational cysteine modification can occur in cells.23,24 Like other cysteine-based redox switches, the sulfenyl amide (300 mM, pH 7) for 2 h gave the thiol derivative 5 in high isolated residue has the potential to undergo ‘overoxidation’ to sulfinyl yield (85%).32 Similar results were obtained when 2 was mixed and sulfonyl derivatives (reactions iv and vii, Scheme 2). Therefore, with thiol in methylene chloride containing catalytic amounts of complete understanding of this redox switch requires consider- triethylamine as a base. These reactions presumably proceed via ation of the reactivity of these higher oxidation states under phys- the thiosulfinate, sulfenic acid, and disulfide intermediates shown iologically relevant conditions. In the work described here we in Scheme 4.33 Consistent with this idea, HPLC analysis at early employed small organic molecules to model the reactivity of pro- times in the reaction between 2 (50 lM) and 2-mercaptoethanol tein sulfinyl and sulfonyl amides that are potential ‘overoxidation’ (500 lM) in sodium phosphate buffer (50 mM, pH 7, containing products of the sulfenyl amide redox switch. Classic25–28 and mod- 30% acetonitrile by volume) revealed an intermediate whose reten- ern29,30 studies have employed model compounds to define the tion time and mass match that of the disulfide 4 at m/z 316 for inherent reactivity of critical functional groups found at enzyme (M+H)+ (Fig. 1). Further, when the reaction was conducted using active sites. Such studies provide insight regarding the chemical only two equivalents of thiol, HPLC analysis showed the disulfide mechanisms of enzyme catalysis and lay a foundation for under- to be a major final product alongside unreacted starting material standing how protein microenvironments alter inherent organic and 5. The thiol, 2-mercaptoethanol, is converted to the corre- reactivities to achieve observed enzyme function. sponding disulfide in this reaction. The present studies build upon our previous use of compound 1 The reaction of 2 (50 lM) with excess thiol (1 mM) in sodium to model the reactivity of the protein sulfenyl amide residue found phosphate buffer (50 mM, pH 7, containing 50% acetonitrile by vol- 22 in PTP1B. For these studies, it may be important that the pKa of ume) occurs with a pseudo-first-order rate constant of 5.5 ± 0.2 Â À3 À1 the corresponding thiol (5) and, thus, the leaving group ability of 10 s (t1/2 2 min). This corresponds to an apparent second-or- the sulfur residue in 1 resembles that of the catalytic cysteine res- der rate constant of 5.5 ± 0.2 MÀ1sÀ1 (Fig. 2). These results offer the idue in PTP enzymes.22 prediction that ‘overoxidation’ of the sulfenyl amide redox switch The ‘overoxidized’ sulfenyl amide model compounds 2 and 3 for to the sulfinyl amide in proteins is a chemically reversible event, be- use in the present studies were prepared by oxidation of 122 with cause the sulfinyl amide can be easily returned to the native cys- dimethyldioxirane (Scheme 3).31 We first examined the reactivity teine thiol residue via reactions with thiols (reaction v, Scheme 2). of the sulfinyl amide 2 with thiol. Incubation of 2 (10 mM) with In the absence of thiol, the sulfinyl amide 2 undergoes a rela- 2-mercaptoethanol (150 mM) in aqueous sodium phosphate buffer tively slow reaction with water to yield the corresponding sulfinic acid derivative 6 in 88% yield (Scheme 5, aqueous sodium phos- phate buffer, 50 mM, pH 7, 12 h, 24C).34 Compound 6 was charac- native cysteine terized as its methyl ester derivative 7 following treatment of the sulfinyl amide sulfinic acid in active PTP1B reaction mixture with excess methyl iodide.35 The hydrolysis of 2 O O O 4 RSH (50 lM) in sodium phosphate buffer (50 mM, pH 7, containing H O NH.. N 2 NH.. 50% acetonitrile by volume) occurs with a pseudo-first-order rate v vi À4 À1 – S SO H constant of 4.5 ± 0.2 10 s , corresponding to a half-life of S 2 2 Â RSH O 26 min (Fig.
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