TK (transketolase) undergoes inactivation during biocatalytic processes due to oxidation, substrate and product inhibition, reactivity of aldehyde substrates, irreversible inactivation at low pH, and dissociation of cofactors. However, the contribution of protein denaturation to each of these mechanisms is not fully understood. The urea-induced reversible denaturations of the apo- and holo-enzyme forms of the homodimeric Escherichia coli TK have been characterized, along with the reconstitution of holo-TK from the apoenzyme and cofactors. An unusual cofactor-bound yet inactive intermediate occurs on both the reconstitution and holo-TK denaturation pathways. The denaturation pathways of the holo- and apoenzymes converge at a second intermediate consisting of a partially denatured apo-homodimer. Preliminary investigation of the denaturation under oxidizing conditions reveals further complexity in the mechanisms of enzyme deactivation that occur under biocatalytic conditions.