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.
Deu
Edgar
E
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720-3206, USA.
Kirsch
Jack F
JF
D001219:Aspartate Aminotransferases / Q000737:chemistry*; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D019791:Guanidine / Q000494:pharmacology; D020035:Holoenzymes / Q000737:chemistry; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D020836:Protein Structure, Quaternary; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000494:pharmacology; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D020035:Holoenzymes; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D014508:Urea; D001219:Aspartate Aminotransferases; D019791:Guanidine; C010627:pyridoxamine phosphate