Abstract
Discovered nearly 70 years ago, the allosteric regulation of Escherichia coli aspartate transcarbamoylase (ATCase) is discussed in every biochemistry textbook. ATCase catalyzes the first step in pyrimidine biosynthesis. Despite extensive research, the mechanism by which this enzyme is regulated by pyrimidine and purine nucleotides has remained elusive. Here, we present a detailed analysis of E. coli ATCase using a combination of biochemical assays, small-angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM), and X-ray crystallography, revealing the mechanism of allosteric communication between nucleotide-binding sites and active sites. We show how the pyrimidine pair, CTP and UTP, synergistically inhibit the enzyme by inducing a quaternary structure that enforces active-site cooperativity. Additionally, we provide the first evidence of how the purine pair, ATP and GTP, drive the enzyme into its most active state by promoting an expanded conformation that allows independent function of the active sites. Our findings resolve longstanding questions in the literature and uncover a novel mechanism by which E. coli ATCase regulates the balance of pyrimidines and purines.