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 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.
Osés-Prieto
Juan A
JA
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA.
Bengoechea-Alonso
Maria T
MT
Artigues
Antonio
A
Iriarte
Ana
A
Martinez-Carrión
Marino
M
D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D021983:Cyclophilins / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000737:chemistry; D051381:Rats; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D010455:Peptides; D011732:Pyridoxal Phosphate; D011392:Proline; D001219:Aspartate Aminotransferases; D021983:Cyclophilins; D019791:Guanidine