Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.

Use of Inosine Monophosphate Dehydrogenase Activity Assay to Determine the Specificity of PARP-1 Inhibitors.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in purine nucleotide biosynthesis. It is responsible for catalyzing the oxidation of inosine monophosphate (IMP) into xanthosine monophosphate (XMP). Concurrently, the cofactor NAD+ is reduced to NADH. Poly(ADP-ribose) polymerase 1 (PARP-1) also utilizes NAD+ as a substrate to synthesize poly(ADP-ribose). It has been demonstrated that inhibition of PARP-1 activity can be an effective cancer therapeutic. However, most PARP-1 inhibitors, including olaparib, were developed as NAD+ analogs. Therefore, these inhibitors likely interfere with other NAD+-dependent pathways such as the one involved in de novo purine metabolism. In this chapter, we describe a method to quantitatively measure IMPDH activity by taking advantage of the autofluorescence of the product NADH. We use this method to analyze the effects of olaparib and non-NAD+-like PARP-1 inhibitor (5F02) on IMPDH activity. We found that olaparib, unlike 5F02, significantly inhibits IMPDH activity in a dose-dependent manner. Our results suggest that IMPDH inhibition is an off-target effect of olaparib treatment. The consequences of this effect should be addressed by future clinical studies.