The biological buffer bicarbonate/CO2 potentiates H2O2-mediated inactivation of protein tyrosine phosphatases.

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 H(2)O(2)-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/CO(2) potentiates the ability of H(2)O(2) 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 H(2)O(2) with HCO(3)(-)/CO(2).

Biologically relevant chemical properties of peroxymonophosphate (=O3POOH).

It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-derived regulator of cellular protein tyrosine phosphatase activity under physiological or pathophysiological conditions. To facilitate further consideration of the potential role of peroxymonophosphate in biological systems we present studies related to the preparation, characterization, stability, and fluorometric detection of this agent.

Oxidative inactivation of protein tyrosine phosphatase 1B by organic hydroperoxides.

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.

Kinetics and mechanism of protein tyrosine phosphatase 1B inactivation by acrolein.

Human cells are exposed to the electrophilic alpha,beta-unsaturated aldehyde acrolein from a variety of sources. The reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well-characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B ( k inact = 0.02 +/- 0.005 s (-1) and K I = 2.3 +/- 0.6 x 10 (-4) M). The enzyme activity does not return upon gel filtration of the inactivated enzyme, and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together, these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon-carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally related alpha,beta-unsaturated aldehydes.