Related Experiment Videos
Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein
Z Y Zhang1, J P Davis, R L Van Etten
1Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393.
Abstract:
Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site-directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pKa values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.
Insights
Covalent modification experiments identified active site residues in 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. Cysteine residues 62 and 145 were labeled, suggesting a histidine-cysteine ion pair in the active site.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- 18-kDa cytoplasmic phosphotyrosyl protein phosphatases are crucial enzymes in cellular signaling.
- Identifying active site residues is key to understanding enzyme function and inhibition.
Purpose of the Study:
- To identify the active site residues of 18-kDa cytoplasmic phosphotyrosyl protein phosphatases using covalent modification.
- To elucidate the mechanism of enzyme inactivation and substrate specificity.
Main Methods:
- Enzyme inactivation using various chemical modifiers (e.g., diethyl pyrocarbonate, phenylglyoxal, iodoacetate, epoxides).
- Analysis of pH dependencies for inactivation reactions.
- Tryptic digestion and sequencing of modified enzyme to identify labeled residues.
Main Results:
- Multiple reagents inactivated the enzyme, indicating modification of active site residues.
- Cysteine residues 62 and 145 were identified as the primary sites of covalent modification by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP).
- Inactivation kinetics and pH dependencies suggest the involvement of reactive cysteine and histidine residues.
Conclusions:
- The active site of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatase likely contains a histidine-cysteine ion pair.
- Substrate specificity is influenced by hydrophobic moieties, as demonstrated by epoxide inactivation.
- Cys-62 and Cys-145 are critical residues for enzyme activity.