Unnatural enzyme activation by a metal-responsive regulatory protein

Abstract

As a result of calcium ion binding, the calcium-dependent regulatory protein calmodulin (CaM) undergoes a conformational change, enabling it to bind to and activate a variety of enzymes. However, the detoxification enzyme glutathione S-transferase (GST) is notably not among the enzymes activated by CaM. In this study, we demonstrate the feasibility of establishing, in vitro, an artificial regulatory link between CaM and GST using bifunctional chemical transducer (CT) molecules possessing binders for CaM and GST. We show that the CTs convert the constitutively active GST into a triggerable enzyme whose activity is unnaturally regulated by the CaM conformational state and consequently, by the level of calcium ions. The ability to reconfigure the regulatory function of CaM demonstrates a novel mode by which CTs could be employed to mediate artificial protein crosstalk, as well as a new means to achieve artificial control of enzyme activity by modulating the coordination of metal ions. Within this study, we also investigated the impact of covalent interaction between the CTs and the enzyme target. This investigation offers further insights into the mechanisms governing the function of CTs and the possibility of rendering them isoform specific.