Intricate systems of regulation at many levels act to control enzyme rates to tune metabolism across biosynthetic pathways. Allostery is a ubiquitous regulatory mechanism for feedback and regulation of enzymes in biosynthetic pathways. ATP-phosphoribosyltransferase from Mycobacterium tuberculosis (mtuATP-PRT) catalyses the first committed step in de novo histidine biosynthesis, and allosteric product inhibition of ATP-PRT by L-His is key to regulating metabolic flux in this pathway. mtuATP-PRT also exhibits allosteric activation by a histidine analogue 3 (2-Thienyl)-L-alanine (TIH). This compound binds in the same allosteric site as L-His and appears to elicit a structurally identical “tensed” conformation, apparently contradicting previous suggestions that the tensed conformation drives inhibitory regulation in mtuATP-PRT. Through mutations adjacent to the allosteric binding site, we have interrupted allosteric inhibition by L-His while retaining allosteric activation by TIH. Kinetic assays of wild type ATP-PRT and mutants across a large temperature range with various allosteric effects, has identified dynamic changes between allosterically modulated states of the enzyme, and suggests an enthalpy-entropy (∆H‡-∆S‡) trade off occurring in allosteric states. Complementing our kinetic data, crystallographic analysis and molecular dynamics (MD) simulations of wild type and mutant ATP-PRT have identified two residues that differ upon the binding of TIH (in comparison to L-His binding). Glu-18 and Arg-27 rotate between ligand bound states and form interacting bonds in L-His bound structures that may serve to stabilise and reduce the overall flexibility of the hexameric conformation. This Glu-18 and Arg-27 interaction varies between L-His-bound, APO-mtuATP-PRT and TIH-bound structures, suggesting this interaction may demarcate allosteric activation and inhibition. An evolutionary analysis of ATP-PRT was undertaken and has identified the long form of ATP-PRT as the ancestral form, and the short form ATP-PRT has subsequently co-opted a histidyl-tRNA synthetase (HisRS) gene product (HisZ) as its regulatory domain. Aminoacyl-tRNA synthetases are known to be prone to horizontal gene transfer, therefore the co-option of HisRS and the loss of the C-terminal domain in ATP-PRTs provides an evolutionary trajectory for diverse ATP-PRT isoforms.