Identification of Hsc70 binding sites in mitochondrial aspartate aminotransferase.

Hsc70 binds acid-unfolded mitochondrial aspartate aminotransferase (mAAT), forming either soluble or insoluble complexes depending on the relative concentrations of the proteins. Using partial proteolysis of Hsc70-mAAT complexes in combination with MALDI-TOF mass spectrometry, we have identified several potential Hsc70-binding regions in the mAAT polypeptide. Only one mAAT peptide was found bound to Hsc70 in the insoluble complexes while nine peptides arising from eight sequence regions of mAAT were found associated with Hsc70 in the soluble complexes.

Electron paramagnetic resonance and fluorescence studies of the conformation of aspartate aminotransferase bound to GroEL.

The interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) with GroEL has been studied by electron paramagnetic resonance (EPR) and fluorescence spectroscopy. In the native protein, the spin probe was immobilized when attached to Cys166 at the domain interface, but was fully mobile when introduced at Cys(-19) in the N-terminal presequence peptide. Unfolding of the protein resulted in a highly mobile EPR spectrum for probes introduced at either site.

The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase.

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.

Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase.

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.

Mitochondrial aspartate aminotransferase catalyses cysteine S-conjugate beta-lyase reactions.

Rat liver mitochondrial aspartate aminotransferase (a homodimer) was shown to catalyse a beta-lyase reaction with three nephrotoxic halogenated cysteine S-conjugates [ S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(2-chloro-1,1,2-trifluoroethyl)-L-cysteine], and less effectively so with a non-toxic cysteine S-conjugate [benzothiazolyl-L-cysteine]. Transamination competes with the beta-lyase reaction, but is not favourable. The ratio of beta elimination to transamination in the presence of S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and 2-oxoglutarate is >100.