Redesigning allosteric activation in an enzyme.

Enzyme activation by monovalent cations is widely documented in plants and the animal world. In type II enzymes, activation entails two steps: binding of the monovalent cation to its allosteric site and transduction of this event into enhanced catalytic activity. The effect has exquisite specificity for either Na(+) or K(+), the most abundant cations present in physiological environments. Enzymes requiring K(+) such as kinases and molecular chaperones are not activated as well or at all by the larger cation Cs(+) or the smaller cations Na(+) and Li(+). Enzymes requiring Na(+) such as β-galactosidase and clotting proteases are not activated as well by Li(+), or the larger cations K(+), Rb(+), and Cs(+). Efforts to switch specificity between Na(+) and K(+) in this large class of enzymes and completely redesign the mechanism of allosteric transduction leading to enhanced catalytic activity have so far been unsuccessful. Here we show how mutagenesis of two loops defining the Na(+) binding site of thrombin, a Na(+)-activated clotting protease, generates a construct that is most active in the presence of K(+) toward synthetic and physiological substrates. The effect is the result of a higher binding affinity and more efficient allosteric transduction of binding into enhanced catalytic activity for K(+) compared to Na(+), which represents a complete reversal of the properties of wild type. In addition, the construct features altered specificity toward physiological substrates resulting in a significant anticoagulant profile. The findings are relevant to all Na(+)-activated proteases involved in blood coagulation and the complement system.