Abstract
Sequential metabolic enzymes have long been hypothesized to form multienzyme metabolic complexes to regulate metabolic flux in cells. Although in vitro biochemistry has not been fruitful to support the hypothesis, advanced biophysical technologies have successfully resurrected the hypothesis with compelling experimental evidence. As biochemistry has always evolved along with technological advancement over the century (e.g., recombinant protein expression, site-directed mutagenesis, advanced spectroscopy and structural biology techniques, etc.), there has been growing interest in advanced imaging-based biophysical methods to explore enzymes inside living cells. In this work, we describe how we visualize two phase-separated biomolecular condensates of multienzyme metabolic complexes that are associated with de novo purine biosynthesis and glucose metabolism in living human cells and how imaging-based data are quantitatively analyzed to advance our knowledge of enzymes and their assemblies in living cells. Therefore, we envision that the framework we describe here would be the starting point to investigate other metabolic enzymes and their assemblies in various cell types with an unprecedented potential to comprehend enzymes and their network in native habitats.