Protein tyrosine phosphatases (PTPs) are important targets of the H(2)O(2) that is produced during mammalian signal transduction. H(2)O(2)-mediated inactivation of PTPs also may be important in various pathophysiological conditions involving oxidative stress. Here we review the chemical and structural biology of redox-regulated PTPs. Reactions of H(2)O(2) with PTPs convert the catalytic cysteine thiol to a sulfenic acid. In PTPs, the initially generated sulfenic acid residues have the potential to undergo secondary reactions with a neighboring amide nitrogen or cysteine thiol residue to yield a sulfenyl amide or disulfide, respectively. The chemical mechanisms by which formation of sulfenyl amide and disulfide linkages can protect the catalytic cysteine residue against irreversible overoxidation to sulfinic and sulfonic oxidation states are described. Due to the propensity for back-door and distal cysteine residues to engage with the active-site cysteine after oxidative inactivation, differences in the structures of the oxidatively inactivated PTPs may stem, to a large degree, from differences in the number and location of cysteine residues surrounding the active site of the enzymes. PTPs with key cysteine residues in structurally similar locations may be expected to share similar mechanisms of oxidative inactivation.
Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H2O2-mediated cellular signal transduction processes but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer, bicarbonate/CO2, potentiates the ability of H2O2 to inactivate PTPs. The results of biochemical experiments and high resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H2O2 with HCO3 −/CO2.
Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively-inactivated PTPs (PTP1B and SHP-2) by various low molecular weight thiols and the enzyme thioredoxin. All thiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rates constants that varied by a factor of approximately eight. In general, molecules bearing low pKa thiol groups were particularly effective. The biological thiol, glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M−1 s−1, while the dithiol, DTT, displayed an apparent second-order rate constant of 0.325 ± 0.007 M−1 s−1. The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 μM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10−3 s−1. The rates at which these agents regenerated oxidized PTP1B followed the trend Trx > DTT > GSH, with comparable values observed at 2 μM Trx, 4 mM DTT and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine (TCEP) regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M−1 s−1. We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.
DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually takes place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the CH/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair.
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