Cofactor-directed reversible denaturation pathways: the cofactor-stabilized Escherichia coli aspartate aminotransferase homodimer unfolds through a pathway that differs from that of the apoenzyme.

While the urea-mediated unfolding pathway of the Escherichia coli aspartate aminotransferase (eAATase) homodimer proceeds through a reversible three-state process with a partially folded dimeric intermediate, D D* 2U (E. Deu and J. F. Kirsch, accompanying paper), that of a cofactor-stabilized form differs. Pyridoxal phosphate, which binds at the intersubunit active sites, stabilizes the native form by 6 kcal mol-1 and dissociates during the D <==> D* transition. Reductive trapping of the cofactor to a nondissociable derivative (PPL-eAATase) precludes the formation of D*. A novel monomeric intermediate (M'-PPL) with 70% of the native secondary structure (circular dichroism) was identified in the unfolding pathway of PPL-eAATase: D-PPL2 <==> 2M'-PPL <==> 2U-PPL. The combined results define two structural regions with distinct stabilities: the active site region (ASR) and the generally more stable, dimerization region (DMR). The DMR includes the key intersubunit contacts. It is responsible for the multimeric nature of D*, and its disorder leads to dimer dissociation. Selective strengthening of the ASR-cofactor interactions by cofactor trapping reverses the relative stabilities of the two regions (from DMR > ASR in the apoenzyme to ASR > DMR in PPL-eAATase) and results in a reordering of the eAATase denaturation pathway.