The exploration provided the first comprehensive mechanistic and kinetic characterization of the 192 uncatalyzed rTCA cycle and its main off-cycle reactions (Fig. ). Intended transition states were 193 obtained for all main cycle reactions (See Fig. for main cycle TSs), meaning that the exploration 194 algorithm successfully mapped a continuous path through potential energy space connecting all 195 For the main cycle carbonyl reduction and CO2 addition reactions, there is potentially some latitude in the specific order and location of the reactions. For example, oxaloacetate (S1) and other intermediates contain multiple distinct carbonyl groups, creating the potential for competing reduction pathways. However, reduction of the non-target carbonyl groups faces substantially higher barriers. The carboxyl reduction in oxaloacetate (R1) requires 92.7 kcal⸱mol -1 compared to the keto group pathway while competing reductions in R17 face minimum barriers ³62.7 kcal⸱mol - 1 . These intrinsic reactivity differences naturally enforce the same carbonyl reduction reaction sequences observed in modern enzymatic pathways, suggesting that chemical kinetics may have guided the evolution of the biological rTCA cycle.