Orange Pyruvate Decarboxylase: Isolation and Mechanistic Studies

Pyruvate decarboxylase, isolated from orange vesicle tissue, was purified 66‐fold by Carbowax®‐4000 and ammonium sulfate precipitations, and carboxymethylcellulose chromatography. The enzyme was characterized for optimum pH, stability, and divalent cation and inhibitor effects. Kinetic studies indicated that the orange enzyme is mechanistically similar to yeast pyruvate decarboxylase, having separate binding sites for Mg ++ and thiamine pyrophosphate. The enzyme differs from its yeast counterpart, in having only one active site. It is specific for pyruvate and 2‐ketobutyrate, and is competitively inhibited by 2‐ketononanoate. The enzyme apparently exists in the fruit, primarily as an inactive cyclizable ternary complex of apo ‐enzyme, Mg ++ , and thiamine pyrophosphate. Cycli‐zation to the active holo‐enzyme is apparently controlled by the available pyruvate concentration.