Both the native L-form and the mirror image D-form of the enzyme 4-oxalocrotonate tautomerase (4OT) were prepared by total chemical synthesis. Our results indicate that both enzymes were efficient catalysts and demonstrate, as expected, that the achiral substrate 2-hydroxymuconate (2) was processed with equal efficiency by either the D- or the L-enzyme. The stereochemical course of the D-4OT-catalyzed reaction in (H2O)-H-2 was also characterized; and it was found that D-40T ketonized 2-hydroxymuconate (2) to (5R)-2-oxo-3(E)-[5-H-2]hexenedioate (3). This finding is consistent with the stereochemical course previously established for the L-4OT-catalyzed reaction and confirms the expectation that the mirror image enzyme molecules D- and L-4OT operate on opposite faces of the dienol intermediate. Furthermore, we have used electrospray ionization time-of-flight (ESI-TOF) mass spectrometry to establish the multimeric state of our synthetic enzymes. Our ESI-TOF results under nondenaturing solution conditions show that each enantiomer formed a noncovalent, homohexameric complex consistent with the previously reported crystallographic analysis of recombinant L-4OT.