We have characterised the early events on the urea denaturation pathway of E coli transketolase, to provide insight into the mechanisms of enzyme deactivation that occur under biocatalytic conditions. Fluorescence intensity and circular dichroism (CD) have revealed three transitions in the denaturation pathway for holo-TK. The first step, at low urea concentration corresponds to the local restructuring of the thiamine diphosphate (TPP) binding-sites. Next, the dissociation of the TPP cofactors and partial loss of secondary structure produces a highly expanded form which is most consistent with a partially denatured dimeric form of the enzyme. The denaturation pathway of apo-TK is significantly less cooperative than that for holo-TK and their pathways converge only after the second transition indicating that the cofactors dissociate before the third and final transition to random coil. These results show that while the enzyme is deactivated initially by changes in structure associated with the cofactors, this event does not release the cofactor from the enzyme. The intermediate state formed is also consistent with that formed during reconstitution of holo-TK from apo-TK. Improvement of biocatalytic processes using TK over prolonged reaction times would, therefore, need to address the formation of this cofactor-associated intermediate state. (C) 2007 Elsevier Inc. All rights reserved.