PMID- 10446379
OWN - NLM
STAT- MEDLINE
DCOM- 19991001
LR  - 20190610
IS  - 0006-3002 (Print)
IS  - 0006-3002 (Linking)
VI  - 1433
IP  - 1-2
DP  - 1999 Aug 17
TI  - Thermal unfolding of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase 
      studied by differential scanning calorimetry.
PG  - 294-306
AB  - 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.
FAU - Levashov, P
AU  - Levashov P
AD  - A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 
      Moscow 119899, Russia.
FAU - Orlov, V
AU  - Orlov V
FAU - Boschi-Muller, S
AU  - Boschi-Muller S
FAU - Talfournier, F
AU  - Talfournier F
FAU - Asryants, R
AU  - Asryants R
FAU - Bulatnikov, I
AU  - Bulatnikov I
FAU - Muronetz, V
AU  - Muronetz V
FAU - Branlant, G
AU  - Branlant G
FAU - Nagradova, N
AU  - Nagradova N
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - Biochim Biophys Acta
JT  - Biochimica et biophysica acta
JID - 0217513
RN  - 0U46U6E8UK (NAD)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
SB  - IM
MH  - Animals
MH  - Calorimetry, Differential Scanning
MH  - Escherichia coli
MH  - Geobacillus stearothermophilus
MH  - Glyceraldehyde-3-Phosphate Dehydrogenases/*chemistry/genetics/isolation & 
      purification
MH  - Muscles/enzymology
MH  - Mutation
MH  - NAD/chemistry/pharmacology
MH  - Protein Conformation/drug effects
MH  - Protein Folding
MH  - Rabbits
MH  - Temperature
EDAT- 1999/08/14 00:00
MHDA- 1999/08/14 00:01
CRDT- 1999/08/14 00:00
PHST- 1999/08/14 00:00 [pubmed]
PHST- 1999/08/14 00:01 [medline]
PHST- 1999/08/14 00:00 [entrez]
AID - S0167-4838(99)00132-6 [pii]
AID - 10.1016/s0167-4838(99)00132-6 [doi]
PST - ppublish
SO  - Biochim Biophys Acta. 1999 Aug 17;1433(1-2):294-306. doi: 
      10.1016/s0167-4838(99)00132-6.