RGD Reference Report - The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase. - Rat Genome Database

Send us a Message



Submit Data |  Help |  Video Tutorials |  News |  Publications |  Download |  REST API |  Citing RGD |  Contact   

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

Authors: Oses-Prieto, JA  Bengoechea-Alonso, MT  Artigues, A  Iriarte, A  Martinez-Carrion, M 
Citation: Oses-Prieto JA, etal., J Biol Chem. 2003 Dec 12;278(50):49988-99. Epub 2003 Sep 30.
RGD ID: 1582383
Pubmed: PMID:14522984   (View Abstract at PubMed)
DOI: DOI:10.1074/jbc.M309922200   (Journal Full-text)

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.

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
amino acid metabolic process  IDA 1582383 RGD 
dicarboxylic acid metabolic process  IDA 1582383 RGD 

Molecular Function
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
amino acid binding  IDA 1582383 RGD 
carboxylic acid binding  IDA 1582383 RGD 
L-aspartate:2-oxoglutarate aminotransferase activity  IDA 1582383 RGD 
pyridoxal phosphate binding  IDA 1582383 RGD 

Objects Annotated

Genes (Rattus norvegicus)
Got2  (glutamic-oxaloacetic transaminase 2)


Additional Information