PROJECT SUMMARY Glucuronidation is often the source of unfavorable pharmacokinetics or pharmacodynamics that lead to the failure of drugs during clinical trials, and as such, there is a critical need in drug development for a tool to control glucuronidation. Our long-term goal is to develop strategies to control glucuronidation by limiting its substrate availability. To do this, we will determine the allosteric mechanism that controls human UDP-glucose dehydrogenase (hUGDH), the enzyme that produces the essential substrate for glucuronidation. In our previous grant, we discovered how the 30-residue intrinsically disordered C-terminus (the ID-tail) modifies the structure of the enzyme to favor binding of the feedback inhibitor UDP-Xyl, a downstream metabolite. We also discovered a cryptic allosteric site for inhibiting the enzyme. Briefly, the feedback inhibitor UDP-Xylose competes with substrate for the active site; upon binding, UDP-Xyl induces the enzyme to slowly isomerize into an inactive complex called EW. The allosteric transition converts the active site into two novel allosteric sites called SBSW and NBSW. The SBSW site is specific for the UDP-Xyl inhibitor, but the NBSW site can bind either UDP-Xyl or the substrate UDP-Glc. We hypothesize that the NBSW and SBSW allosteric sites cooperatively stabilize EW, and the dual-specificity of the NBSW is an important feature that allows the abundant substrate UDP-Glc to enhance the binding affinity of the less abundant inhibitor UDP-Xyl in the SBSW. This hypothesis is based on our preliminary data that (i) the substrate UDP-Glc can bind to the NBSW site and inhibit hUGDH, and (ii) the inhibitor UDP-Xyl can bind to both the SBSW and NBSW to inhibit. This hypothesis will be tested by the following specific aims: 1) we will determine how the NBSW and SBSW allosteric sites interact to enhance the allosteric inhibition by UDP-Xyl; 2) we will determine the relationship between a putative low barrier hydrogen bond (LBHB) and the stability of the NBSW and SBSW allosteric sites; and 3) we will identify the structural features that couple the intrinsically disordered C-terminus (ID-tail) of hUGDH to the favorable formation of EW. The research proposed in this application is innovative because it focuses on the allosteric inhibition of hUGDH as a global mechanism for controlling glucuronidation, and uses our recent discoveries of: (i) the novel NBSW allosteric site; (ii) a putitive low barrier hydrogen bond in the allosteric mechanism; and (iii) the entropic force generated by the intrinsically disordered C-terminus. Since these features are recent discoveries from my lab, this research is distinct from previous attempts that tried to control glucuronidation. The expected outcomes of this work are significant. A detailed description of the allosteric mechanism of hUGDH will serve as a foundation for the design of a class of allosteric inhibitors that will act as global regulators of glucuronidation. And more...