Abstract
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 noncatalytic residue in the WPD-loops of YopH and PTP1B result in shifted pH-rate profiles from an altered kinetic pKa of the nucleophilic cysteine. Compared to wild type, 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 nonchemical 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.
Highlights
Enzymatic activity is usually pH-dependent, and this behavior is one of the standard parameters measured during in vitro kinetics studies of enzymes to identify catalytic residues and mechanisms
Structures were obtained of the PTP1B T177G and YopH G352T ligand-free enzymes and in complex with vanadate
Comparison of the variant structures with those of the native enzymes show no significant backbone perturbations in the WPD-loop regions or elsewhere (Figure 2). In both ligand-free structures, the WPD-loops are observed in the closed position (Figure 3). This is in contrast to the typical observation of this loop in the open, noncatalytic position in crystal structures of these and other protein tyrosine phosphatases (PTPs) lacking a bound oxyanion
Summary
Enzymatic activity is usually pH-dependent, and this behavior is one of the standard parameters measured during in vitro kinetics studies of enzymes to identify catalytic residues and mechanisms. This factor has a more fundamental importance in biology, where an enzyme’s biological activity is affected by the pH of its microenvironment. This pH may or may not correspond to the optimum found in laboratory kinetics studies and can vary depending on the organism, within different compartments of the cell or cell type, and is different in healthy cells compared to the more acidic conditions within tumor microenvironments.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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
