Examination of the Transition State of the Low-Molecular Mass Small Tyrosine Phosphatase 1. Comparisons with Other Protein Phosphatases

Authors

Alvan C. Hengge, Utah State UniversityFollow
Y. Zhao
L. Wu
Z. Y. Zhang

Document Type

Article

Journal/Book Title

Biochemistry

Publication Date

1997

Volume

36

First Page

7928

Last Page

7936

Abstract

The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [¹⁸(V/K)_nonbridge], at the bridging oxygen atom (the site of bond cleavage) [¹⁸(V/K)_bridge], and at the nitrogen atom in the nitrophenol leaving group [¹⁵(V/K)]. The results with native Stp1 were 1.0160 ± 0.0005 for ¹⁸(V/K)_bridge, 1.0007 ± 0.0001 for ¹⁵(V/K), and 1.0018 ± 0.0003 for ¹⁸(V/K)_nonbridge. The values for ¹⁸(V/K)_nonbridge and ¹⁵(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P−O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for ¹⁸(V/K)_bridge of about 1.029, for ¹⁵(V/K) of about 1.003, and for ¹⁸(V/K)_nonbridge of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

Recommended Citation

A. C. Hengge, Y. Zhao, L. Wu, Z.-Y. Zhang. “Examination of the Transition State of the Low- Molecular Mass Small Tyrosine Phosphatase 1. Comparisons with Other Protein Phosphatases.” Biochemistry, 1997, 36, 7928-7936.