Unfolded, oxidized, and thermoinactivated forms of glyceraldehyde-3-phosphate dehydrogenase interact with the chaperonin GroEL in different ways

Abstract

The interaction of GroEL with different denatured forms of glyceraldehyde-3-phosphate dehydrogenase* (GAPDH) has been investigated. GroEL does not prevent thermal denaturation of GAPDH, but effectively interacts with the thermodenatured enzyme, thus preventing the aggregation of denatured molecules. Binding of the thermodenatured GAPDH shifts the T m value of the GroEL thermodenaturation curve by 3° towards higher temperatures and increases the Δ H cal value 1.44-fold, indicating a significant increase in the thermal stability of the resulting complex. GAPDH thermodenatured in the presence of GroEL cannot be reactivated by the addition of GroES, Mg 2+, and ATP. In contrast, GAPDH denatured in guanidine hydrochloride (GAPDH den) is reactivated in the presence of GroEL, GroES, Mg 2+, and ATP, yielding 11–15% of its original activity, while the spontaneous reactivation yields only 2–3%. The oxidation of GAPDH with hydrogen peroxide in the presence of 4 M guanidine hydrochloride results in the formation of the enzyme (GAPDH ox) that cannot acquire its native conformation and binds to GroEL irreversibly. Binding of GAPDH ox to one of the GroEL rings completely inhibits the GroEL-assisted reactivation of GAPDH den, but does not affect the GroEL-assisted reactivation of lactate dehydrogenase (LDH). The data suggest that LDH can be successfully reactivated due to the binding of the denatured molecules to the apical domain of the opposite GroEL ring with their subsequent release into the solution without encapsulation ( trans-mechanism). In contrast, GAPDH requires the hydrophilic cavity for the reactivation ( cis-mechanism).