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Pyruvate carboxylase (PC) is a biotin-dependent enzyme essential for anaplerotic flux into the tricarboxylic acid cycle and gluconeogenesis. Despite longstanding biochemical knowledge, the structural mechanisms governing its allosteric regulation and domain coordination remain poorly defined. Here, we present the cryo-EM structures of Mycobacterium tuberculosis PC (MtPC) captured in both apo and substrate-bound forms. In the absence of acetyl-CoA, MtPC forms a flexible and unstable tetramer that readily dissociates into subcomplexes. Upon binding of acetyl-CoA and the substrates, the enzyme transitions into a compact, ordered assembly, with acetyl-CoA acting as a molecular clamp that stabilizes inter-subunit interactions. Remarkably, we captured the mobile biotinylated domain simultaneously engaging each catalytic center within monomers, providing direct structural evidence for the two steps of the catalytic cycle. 3D variability analysis further revealed how this mobile domain translocates between the active sites during catalysis. Together, these findings demonstrate how acetyl-CoA stabilizes MtPC by reinforcing inter-subunit contacts and elucidate the conformational dynamics underlying domain coordination during catalysis. These findings provide new structural insights into the allosteric regulation of this essential metabolic enzyme.