Oxidative inactivation of protein tyrosine phosphatase 1B by organic hydroperoxides

Abstract

Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell signaling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a manner that mirrors the endogenous signaling agent hydrogen peroxide.Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that catalyze the hydrolytic removal of phosphate groups from tyrosine residues in proteins (Scheme 1). [1][2][3] Thus, PTPs, in concert with protein tyrosine kinases, play a central role in cell signaling by regulating the phosphorylation status and, in turn, the functional properties, of target proteins in various signal transduction pathways. 4,5 The cellular activity of some PTPs is regulated by hydrogen peroxide (H 2 O 2 ) that is produced as a second messenger in response to extracellular stimuli such as insulin, epidermal growth factor, and platelet derived growth factor. 6-9 H 2 O 2 inactivates PTPs via oxidation of the active site cysteine thiol residue to a sulfenic acid. 6,10 In some cases, the cysteine sulfenic acid undergoes subsequent conversion to an active site sulfenyl amide linkage 11-13 or disulfide. 14,15 This type of oxidative inactivation of PTPs is slowly reversed upon reaction of the inactivated enzyme with biological thiols (Scheme 1). [10][11][12][13][14][15] Given the biological importance of PTPs, it may be useful to identify organic reagents that regulate PTP activity through redox mechanisms. [16][17][18][19][20] With this in mind, we examined the ability of several organic hydroperoxides (1-4) to effect thiol-reversible, oxidative inactivation of the archtypal member of the PTP family, PTP1B. 21 We find that peracetic acid (1) is a potent, time-dependent inactivator of the catalytic subunit of PTP1B (a.a. 1-322) (Fig. 1A). The observation of time-dependent inactivation is consistent with a process involving covalent modification of the enzyme. 22 A good linear fit is obtained in a plot of the apparent rates of inactivation versus concentration indicating that the inactivation reaction is a second-order process (Fig. 1B). The slope of the line reveals a rate constant of 2.1 ± 0.2 × 10 4 M −1 s −1 for the reaction of 1 with PTP1B. For comparison, hydrogen peroxide inactivates PTP1B with a rate constant of 10 M −1 s −1 . 10 The inactivation of PTP1B (250 nM) by 1 (1 μM, 3 min) is not reversed upon removal of excess inactivator by gel filtration of the enzyme through G25 Sephadex. The inactivation process is significantly slowed in the presence of phosphate ion, which is an active site-directed inhibitor of the enzyme (K i = 17 mM). Specifically, treatment of the enzyme with 1 (15 μM) for 15 s inactivates 88% of the enzyme under standard conditions, while in the presence of phosphate *Corresponding author. Tel.: +1-573-882-6763; fax: +1-573-882-2754 gatesk@missouri.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ion (50 mM) only 49% inactivation is observed. Together, the results suggest that 1 inactiv...

An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP

“…The slope gave a second-order rate constant (k inact ) of 185 Ϯ 11 M Ϫ1 min Ϫ1 . This constant is close to values obtained for other enzymes known to be inhibited by reversible oxidation of cysteine residues by H 2 O 2 , such as human arylamine N-acetyltransferase 1 (29), caspase 3 (30), protein tyrosine phosphatases (31,32), or calcineurin (33).…”

Inactivation of Protein Tyrosine Phosphatases by Peracids Correlates with the Hydrocarbon Chain Length

“…5B). Previous studies indicated that organic peroxides, such as peracetic acid, induce reversible inactivation of PTP1B phosphatase [21]. According to Bhattacharya et al [21], reversible PTP oxidation may be a general mechanism of enzymatic inactivation by organic peroxides.…”

“…Previous studies indicated that organic peroxides, such as peracetic acid, induce reversible inactivation of PTP1B phosphatase [21]. According to Bhattacharya et al [21], reversible PTP oxidation may be a general mechanism of enzymatic inactivation by organic peroxides. The results of this study clearly show that the reversible oxidation is not observed for the medium-chain peracids, possibly because their optimal size and structure allows for effective penetration into the catalytic center of CD45 repeatedly, leading to further oxidation of the catalytic cysteine to the irreversible sulfinic and sulfonic acid derivatives.…”

“…It had previously been found that peroxidized arachidonic acid can induce oxidation of PTPs [37]. Another study has shown that peracetic acid is a potent oxidative inhibitor of PTP1B phosphatase [21]. The peroxycarboxyl group (-COOOH) in a peracid is an oxidized derivative of the regular carboxyl group (-COOH) [18].…”

“…It has been shown that peracetic acid (C2-peracid), likely formed in chronic alcoholics from acetic acid and hydrogen peroxide, may inhibit enzyme that contains thiol groups, arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase, localized in the erythrocyte membrane [20]. Another study has shown that peracetic acid (C2-peracid) is a potent oxidative inhibitor of PTP1B phosphatase [21]. In our previous work, we demonstrated that activity of phosphatase CD45 can be decreased by treatment with tetradecanoic peracid [22].…”