Thermal unfolding of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry

Thermal unfolding parameters were determined for a two-domain tetrameric en zyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a nd for its isolated NAD(+)-binding domain. At pH 8.0, the transition temper atures (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 (Delta Hcal) of 4415 and 437 kJ/mol (or 30.7 and 22 .1 J/g), respectively. In the presence of nearly saturating NAD(+) concentr ations, the t(max) and the Delta H-cal increased by 13.6 degrees C and by 2 365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C an d 109 kJ/mol for the isolated domain. These results indicate that interdoma in interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolate d NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native CAPDH exhibited greater stability at pH 6.0; si milar pH-dependencies of thermal stability were displayed by GAPDHs isolate d from rabbit muscle and Escherichia coli. The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and Delta H-cal and diminished the widths of the DSC curves; the effect was found to grow progressively with increas ing coenzyme concentrations. Alkylation of the essential Cys149 with iodoac etamide destabilized the apoenzyme and altered the effect of NAD(+). Replac ement of Cys149 by Ser or by Ala in the B. stearothermophilus CAPDH produce d 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 interact ion between Cys149 and NAD(+) make any significant contribution to the stab ilization of the enzyme's native tertiary structure and the accomplishment of NAD(+)-induced conformational changes. The H176N mutant exhibited dramat ically lower heat stability, as reflected in the values of both Delta H-cal and t(max). Interestingly, NAD(+) binding resulted in much wider heat capa city curves, suggesting diminished cooperativity of the unfolding transitio n. (C) 1999 Elsevier Science B,V. All rights reserved.