The enzyme Threonine Deaminase 2 (TD2) evolved a novel defensive function in tomato after duplication and prolonged adaptive evolution over 30-50 million years: it depletes essential amino acid Threonine in the guts of Lepidoptera larvae. Previous researchers suggested that three novel, TD2-specific critical ion-pair-making substitutions contributed to producing this novel function. We examined the roles of these ion pairs by expressing TD2 variant enzymes with and without these substitutions in E. coli. Kinetic assays demonstrated epistasis: adding any one or two ion pairs had no effect, but adding all three increases catalytic efficiency, which is presumably adaptive because increased catalytic activity likely reduces Threonine absorption. The TD phylogeny supports the inference of adaptation: one ion pair arose long after duplication, a second arose after a similarly long wait, but the third ion pair arose quickly after the addition of the second. This pattern suggests that fitness epistasis prolonged the evolution of catalytic efficiency. The need for permissive substitutions, indicated by the failure of introducing the ion pairs into other TD copies in other species to increase catalytic activity, suggests that the requirement of such substitutions may also have contributed to the prolonged period of adaptive evolution in tomato TD2.