Aspartate transcarbamoylase (ATCase) from Escherichia coli has served as a classic model of enzyme allostery for nearly 70 years. Structurally, ATCase is a heterododecamer, comprising two catalytic trimers that bind substrates cooperatively, as well as three regulatory dimers that bind nucleotides. Despite extensive study, the molecular mechanism of allosteric regulation by nucleotides has remained unresolved. Here, we utilize a combination of activity assays, small-angle X-ray scattering, cryogenic electron microscopy, and crystallography to elucidate this mechanism. As shown in previous work, our findings confirm that substrate binding induces the transition from the compact, inactive T-state to the expanded, active R-state. However, our results reveal that the quaternary structure of the R-state in solution is further modulated by nucleotides. Remarkably, we also demonstrate that ATP and GTP together expand the enzyme even in the absence of substrates, disabling the cooperativity for substrate binding. This observation uncovers a sophisticated regulatory mechanism that deviates from the classical Monod-Wyman-Changeux (MWC) model. Our study thus provides the first comprehensive understanding of nucleotide-driven allosteric regulation in E. coli ATCase.