Abstract
Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal structures of apo-PTP1B, the Michaelis complex of an inactive mutant, the phosphoenzyme intermediate, and the product complex, a full picture of all catalytic steps can now be depicted. The transition state analog for the first catalytic step comprises a ternary complex between the catalytic cysteine of PTP1B, vanadate, and the peptide DADEYL, a fragment of a physiological substrate. The equatorial vanadate oxygen atoms bind to the P-loop, and the apical positions are occupied by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur atom. The vanadate assumes a trigonal bipyramidal geometry in both transition state analog structures, with very similar apical O-O distances, denoting similar transition states for both phosphoryl transfer steps. Detailed interactions between the flanking peptide and the enzyme are discussed.
Highlights
The phosphorylation of tyrosine residues by protein-tyrosine kinases and the reverse action by protein-tyrosine phosphatases (PTPs)4 is a common mechanism for the control of biological pathways [1,2,3]
As the enzymatic substrate of PTPs is the dianionic form of p-nitrophenyl phosphate (pNPP), the 18(V/ K)nonbridge isotope effect was corrected considering the isotope fractionation for protonation of the nonbridge oxygen atoms because pNPP is present as a mixture of monoanion and dianion forms at this pH
Crystallography—The two transition state analogs were crystallized under similar conditions, except for the concentrations of peptide and vanadate, which were optimized to give the structures of the transition state analog for the first step (TSA1) and the second step (TSA2)
Summary
The phosphorylation of tyrosine residues by protein-tyrosine kinases and the reverse action by protein-tyrosine phosphatases (PTPs) is a common mechanism for the control of biological pathways [1,2,3]. A common strategy is focused on the synthesis of competitive inhibitors that block the active site [6, 11, 14] Binding affinities in these cases range from millimolar to micromolar, and specificity is often compromised by the high similarity between PTP active sites. In PTP1B, targeting of a second Tyr(P) binding site in the vicinity of the active site has been used In such cases, inhibition constants can be improved to the nanomolar range [15], and selectivity is gained because the second Tyr(P) site in PTP1B is not conserved among all PTPs. One important reason for the limited success of single site inhibition in PTPs is the fact that substrate binding is far surpassed by the affinity of the TS. Besides the known closure of the WPD-loop, the existence of other catalysis-associated conformational changes that might affect protein-substrate interactions is uncertain
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
