Dissecting the catalytic mechanism of protein-tyrosine phosphatases.
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Journal: 1994/April - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 8127855
Abstract:
Protein-tyrosine phosphatases (PTPases) contain an evolutionarily conserved segment of 250 amino acids referred to as the PTPase catalytic domain. The recombinant PTPase domain from Yersinia enterocolitica enhances the rate of hydrolysis of p-nitrophenyl phosphate, a phosphate monoester, by approximately 10(11) over the non-enzyme-catalyzed rate by water. Specific amino acid residues responsible for the catalytic rate acceleration have been examined by site-directed mutagenesis. Our results suggest that Asp-356 (D356) and Glu-290 (E290) are the general acid and the general base catalysts responsible for Yersinia PTPase-catalyzed phosphate ester hydrolysis. The PTPase with both E290Q and D356N mutations shows no pH dependence for catalysis but displays a rate enhancement of 2.6 x 10(6), compared to the noncatalyzed hydrolysis of p-nitrophenyl phosphate by water. This rate enhancement probably occurs via transition-state stabilization. Our results suggest that all PTP-ases use a common mechanism that depends upon formation of a thiol-phosphate intermediate and general acid-general base catalysis.
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Proc Natl Acad Sci U S A 91(5): 1624-1627
PMID: 8127855

Dissecting the catalytic mechanism of protein-tyrosine phosphatases.

Abstract

Protein-tyrosine phosphatases (PTPases) contain an evolutionarily conserved segment of 250 amino acids referred to as the PTPase catalytic domain. The recombinant PTPase domain from Yersinia enterocolitica enhances the rate of hydrolysis of p-nitrophenyl phosphate, a phosphate monoester, by approximately 10(11) over the non-enzyme-catalyzed rate by water. Specific amino acid residues responsible for the catalytic rate acceleration have been examined by site-directed mutagenesis. Our results suggest that Asp-356 (D356) and Glu-290 (E290) are the general acid and the general base catalysts responsible for Yersinia PTPase-catalyzed phosphate ester hydrolysis. The PTPase with both E290Q and D356N mutations shows no pH dependence for catalysis but displays a rate enhancement of 2.6 x 10(6), compared to the noncatalyzed hydrolysis of p-nitrophenyl phosphate by water. This rate enhancement probably occurs via transition-state stabilization. Our results suggest that all PTP-ases use a common mechanism that depends upon formation of a thiol-phosphate intermediate and general acid-general base catalysis.

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Selected References

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Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
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Abstract
Protein-tyrosine phosphatases (PTPases) contain an evolutionarily conserved segment of 250 amino acids referred to as the PTPase catalytic domain. The recombinant PTPase domain from Yersinia enterocolitica enhances the rate of hydrolysis of p-nitrophenyl phosphate, a phosphate monoester, by approximately 10(11) over the non-enzyme-catalyzed rate by water. Specific amino acid residues responsible for the catalytic rate acceleration have been examined by site-directed mutagenesis. Our results suggest that Asp-356 (D356) and Glu-290 (E290) are the general acid and the general base catalysts responsible for Yersinia PTPase-catalyzed phosphate ester hydrolysis. The PTPase with both E290Q and D356N mutations shows no pH dependence for catalysis but displays a rate enhancement of 2.6 x 10(6), compared to the noncatalyzed hydrolysis of p-nitrophenyl phosphate by water. This rate enhancement probably occurs via transition-state stabilization. Our results suggest that all PTP-ases use a common mechanism that depends upon formation of a thiol-phosphate intermediate and general acid-general base catalysis.
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