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A Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases

preprint
submitted on 18.01.2021, 22:21 and posted on 20.01.2021, 07:50 by Ruidan Shen, Rory Crean, Sean Johnson, Shina Caroline Lynn Kamerlin, Alvan C. Hengge

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic pKa of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.

History

Email Address of Submitting Author

lynn.kamerlin@kemi.uu.se

Institution

Uppsala University

Country

Sweden

ORCID For Submitting Author

0000-0002-3190-1173

Declaration of Conflict of Interest

No conflict of interest.

Version Notes

Version 1.0, initial submission.

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ChemRxiv

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