Barbiturase consists of four identical subunits, each bound to a zinc (Zn) atom.
Absorption spectrum analysis illustrates that zinc is the only cofactor present in barbiturase.
Several highly conserved histidine residues were found in the zinc binding motif region of barbiturase, suggesting that histidine residues are involved in zinc binding and are necessary for the catalytic activity of barbiturase.
Finally, barbiturase activity can be blocked upon addition of other metal ions, such as copper and mercury.
In addition, barbituric acid inhibits multiple enzymes that are involved in de novo pyrimidine synthesis.