title: Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase.
abstract: Dimeric mitochondrial aspartate aminotransferase (mAAT) contains a molecule of pyridoxal 5'-phosphate (PLP) tightly attached to each of its two identical active sites. The presence of this natural reporter allows us to study separately local perturbations in the architecture of this critical region of the molecule during unfolding. Upon unfolding of the enzyme with guanidine hydrochloride (GdnHCl), the coenzyme is completely released from the active site. The transition midpoint for the dissociation of PLP is 1.4+/-0.02 M when determined by size-exclusion chromatography (SEC) and 1.6+/-0.02 M when the protein-bound PLP is estimated by electrospray mass spectrometry (ESI-MS). In both cases the transition midpoint is higher than that of inactivation (1.3+/-0.01 M). On the other hand, the midpoint of the unfolding transition obtained by monitoring changes in ellipticity at 356 nm, which reflects the asymmetric environment of the PLP cofactor at the active site, is 1.19+/-0.011 M guanidine. These results indicate that the unfolding of mAAT is a multi-step process which includes an intermediate containing bound PLP but lacking catalytic activity.
authors.affiliation: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Rd., Kansas City, MO 64110-2499, USA.
authors.forename: Ting-Huai
authors.initials: TH
authors.lastname: Wu
authors.forename: Juan A
authors.initials: JA
authors.lastname: Oses-Prieto
authors.forename: Ana
authors.initials: A
authors.lastname: Iriarte
authors.forename: Marino
authors.initials: M
authors.lastname: Martinez-Carrion
mesh_terms: D000818:Animals; D024442:Aspartate Aminotransferase, Mitochondrial / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D051381:Rats
chemical_list: D011732:Pyridoxal Phosphate; D024442:Aspartate Aminotransferase, Mitochondrial