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Oxidative inactivation of the lymphoid tyrosine phosphatase mediated by both general and active site directed NO
Caitlin E Karver1, Vanessa F Ahmed, Amy M Barrios
1Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, United States.
Abstract:
Oxidative modification of protein tyrosine phosphatases (PTPs) has recently been recognized as an important regulatory mechanism in biological systems. Reported herein is the oxidative inactivation of the lymphoid tyrosine phosphatase (LYP) with both the general nitrosating reagent sodium nitroprusside (SNP) and also a novel peptide-based nitrosating reagent, Ac-ARLIEDNE(HcyNO)TAREG-NH(2), where HcyNO = S-nitrosohomocysteine. The SNP oxidatively inactivated LYP with a k(inact) of 0.383 per min and a K(I) of 27.4 μM and mixed-type inactivation kinetics. The peptide was a competitive LYP inactivator with a k(inact) of 0.0472 per min and a K(I) of 7.00 μM. LYP nitrosation by SNP was characterized by the addition of several NO moieties to the enzyme, while oxidation of LYP by the peptide did not result in the formation of a LYP-NO adduct. We propose that general NO donors promiscuously nitrosate any free cysteine residue while the active-site directed peptide selectively oxidizes the catalytic cysteine residue, resulting in the formation of a disulfide bond between the catalytic cysteine residue and a second cysteine in the active site.
Insights
Oxidative modification of protein tyrosine phosphatases (PTPs) regulates biological systems. Researchers found that the lymphoid tyrosine phosphatase (LYP) is inactivated by nitrosating reagents, with a novel peptide selectively targeting its active site.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Oxidative modification of protein tyrosine phosphatases (PTPs) is a critical regulatory mechanism.
- Lymphoid tyrosine phosphatase (LYP) plays a role in biological signaling pathways.
Purpose of the Study:
- To investigate the oxidative inactivation of LYP using different nitrosating reagents.
- To elucidate the mechanism of LYP inactivation by sodium nitroprusside (SNP) and a novel peptide-based reagent.
Main Methods:
- Enzyme kinetics studies using SNP and a synthesized S-nitrosohomocysteine peptide.
- Characterization of LYP inactivation kinetics and identification of modification sites.
Main Results:
- SNP caused mixed-type inactivation of LYP (k(inact) = 0.383/min, K(I) = 27.4 μM), involving multiple NO additions.
- The peptide reagent competitively inactivated LYP (k(inact) = 0.0472/min, K(I) = 7.00 μM) without forming a LYP-NO adduct.
- SNP nitrosates free cysteines, while the peptide selectively oxidizes the catalytic cysteine.
Conclusions:
- LYP inactivation mechanisms differ between general NO donors and active-site directed reagents.
- The peptide-based reagent offers a selective method for studying LYP oxidation.
- Oxidative modification of LYP, particularly at the catalytic cysteine, is a key regulatory event.
