Thermal unfolding of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry.
Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD(+)-binding domain. At pH 8.0, the transition temperatures (t(max)) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3 degrees C and 61.9 degrees C, with calorimetric enthalpies (DeltaH(cal)) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively. In the presence of nearly saturating NAD(+) concentrations, the t(max) and the DeltaH(cal) increased by 13.6 degrees C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C and 109 kJ/mol for the isolated domain. These results indicate that interdomain interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolated NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native GAPDH exhibited greater stability at pH 6.0; similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli. The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and DeltaH(cal) and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations. Alkylation of the essential Cys149 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD(+). Replacement of Cys149 by Ser or by Ala in the B. stearothermophilus GAPDH produced some stabilization, the effect of added NAD(+) being basically similar to that observed with the wild-type enzyme. These data indicate that neither the ion pairing between Cys149 and His176 nor the charge transfer interaction between Cys149 and NAD(+) make any significant contribution to the stabilization of the enzyme's native tertiary structure and the accomplishment of NAD(+)-induced conformational changes. The H176N mutant exhibited dramatically lower heat stability, as reflected in the values of both DeltaH(cal) and t(max). Interestingly, NAD(+) binding resulted in much wider heat capacity curves, suggesting diminished cooperativity of the unfolding transition.
Levashov
P
P
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
Orlov
V
V
Boschi-Muller
S
S
Talfournier
F
F
Asryants
R
R
Bulatnikov
I
I
Muronetz
V
V
Branlant
G
G
Nagradova
N
N
D000818:Animals; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli; D001411:Geobacillus stearothermophilus; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D009243:NAD / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011817:Rabbits; D013696:Temperature
D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases