Unfolded, oxidized, and thermoinactivated forms of glyceraldehyde-3-phosphate dehydrogenase interact with the chaperonin GroEL in different ways.
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 Tm value of the GroEL thermodenaturation curve by 3 degrees towards higher temperatures and increases the DeltaHcal 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, Mg2+, and ATP. In contrast, GAPDH denatured in guanidine hydrochloride (GAPDHden) is reactivated in the presence of GroEL, GroES, Mg2+, 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 (GAPDHox) that cannot acquire its native conformation and binds to GroEL irreversibly. Binding of GAPDHox to one of the GroEL rings completely inhibits the GroEL-assisted reactivation of GAPDHden, 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).
School of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskie gory, Moscow 119992, Russia.
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Naletova
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Muronetz
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Schmalhausen
D000818:Animals; D002152:Calorimetry, Differential Scanning; D018834:Chaperonin 60 / Q000737:chemistry*; D004789:Enzyme Activation; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry*; D019791:Guanidine; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000737:chemistry; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D009132:Muscles / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits
D018834:Chaperonin 60; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D019791:Guanidine