TPI has been found in nearly every organism searched for the enzyme, including animals such as mammals and insects as well as in fungi, plants, and bacteria.

[1] Triose phosphate isomerase is a highly efficient enzyme, performing the reaction billions of times faster than it would occur naturally in solution.

The reaction is so efficient that it is said to be catalytically perfect: It is limited only by the rate the substrate can diffuse into and out of the enzyme's active site.

Besides the precisely placed glutamate and histidine residues to form the enediol, a ten- or eleven-amino acid chain of TPI acts as a loop to stabilize the intermediate.

[7] In addition to making the reaction kinetically feasible, the TPI loop sequesters the reactive enediol intermediate to prevent decomposition to methylglyoxal and inorganic phosphate.

The hydrogen bond between the enzyme and the phosphate group of the substrate makes such decomposition stereoelectronically unfavorable.

[11] The loss of a high-energy phosphate bond and the substrate for the rest of glycolysis makes formation of methylglyoxal inefficient.

Studies suggest that a lysine close to the active site (at position 12) is also crucial for enzyme function.