Coupling of Loop Closure and Chemistry in Protein Tyrosine Phosphatases

Abstract

Protein tyrosine phosphatases (PTPs) are a class of enzymes that catalyze the dephosphorylation of tyrosine residues. PTPs share a common fold with an active site geometry consisting of two loops a P-loop that is responsible for phosphate binding and a WPD-loop, named for its consensus WPD sequence, which includes a critical Asp residue that functions as a general acid/base during catalysis. Comparison of uncomplexed crystal structures with those bound to oxyanion ligands shows that the WPD-loop is flexible, alternating between “open” and “closed” conformations. In the closed form, the catalytic Asp residue is within hydrogen bonding distance of the oxyanion; loop closure is essential for catalysis.Two PTPs studied here are YopH a critical virulence factor from Yersinia and human PTP1B. Each enzyme possesses homologous WPD loops and the chemical step is fully rate limiting yet surprisingly their kcat values differ by an order of magnitude, with YopH being 10-fold faster. Using NMR spectroscopy (at pH=6.5, 293K) we have addressed the role of loop motion in the catalytic activity of these two PTPs. The first of these, PTP1B is a less powerful catalyst than YopH with kcat ∼ 17s−1. NMR CPMG relaxation dispersion studies remarkably show that the WPD loop in apo PTP1B closes with kclose = 17 ± 8 s−1. YopH catalyzes phosphate hydrolysis with a kcat ∼100 - 200 s−1. Estimates of the WPD loop exchange rate constant, were made from from Hahn echo experiments and 1HN-R1ρ dispersion profiles that suggest kclose = 400 ± 200 s–1.Thus, differences in loop closing rates closely correlates with the respective kcat values in these two enzymes and further suggests a role for the frequency of WPD-loop motion in the catalytic function of protein tyrosine phosphatases.