The Nature of the Rate-limiting Steps in the Refolding of the Cofactor-dependent Protein Aspartate Aminotransferase

Abstract

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 equilibriumunfolded 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 Ϸ 0.75 min ؊1 at 20°C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stoppedflow 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 Ϸ 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.The folding and unfolding of proteins are complex processes affected by a number of features such as size, chain topology, and oligomeric state and ultimately by the amino acid sequence, which encodes each unique three-dimensional structure (1). Much of our understanding of the protein folding problem arises from in vitro refolding studies of small monomeric proteins (2). However, the molecular mass of the majority of proteins found in prokaryotic or eukaryotic cells is larger than 25 kDa (3). Most of these larger polypeptides fold into several structural domains, and in the case of oligomeric proteins several subunits associate through noncovalent interactions. Folding and unfolding of many large proteins with several folding domains and/or multiple subunits often show multiphasic kinetics with fast and slow folding phases, suggesting the presence of either multiple paths or intermediate states in the process (4). Slow folding phases are often associated with proline isomerization (5, 6), but in some cases the rate-limiting step involves, at least to some extent, the reorganization of misfolded species or metastable kinetic folding intermediates (7-9). The significance of these kinetic intermediates is still a matter of discussion. They are considered to represent either productive intermediates that facilitate folding or kinet...

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: Thermodynamic and crystallographic studies

“…In other words, the recorded differences seem to suggest a possible dependence on the cofactor of the unfolding kinetics and the formation of unfolded species. Similar suggestions have been put forward for AAT 41…”

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

“…Our results are in good agreement with those of NMR studies of the reaction between PLP and free lysine, which led Chan-Huot et al to conclude that, at pH values below 4, PLP no longer forms a Schiff base with lysine, but rather exists as a free hydrate (44). Moreover, time-dependent dissociation of PLP has been reported for the guanidine deuterochloride denaturation of aspartate aminotransferase (45). …”

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

“…The effect of PLP on folding varies among the different PLP-dependent enzymes analysed to date. The isolated β 2 subunit of tryptophan synthase [43] and the cytosolic aspartate aminotransferase [44] require a coenzyme for re-activation in vitro, whereas refolding of E. coli serine hydroxymethyltransferase [45] and mitochondrial aspartate aminotransferase [46] can proceed in the absence of coenzyme. The dependence of the refolding yield on MalY concentration was also examined.…”