PMID- 12056886
OWN - NLM
STAT- MEDLINE
DCOM- 20020726
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 41
IP  - 24
DP  - 2002 Jun 18
TI  - Thermal unfolding used as a probe to characterize the intra- and intersubunit 
      stabilizing interactions in phosphorylating D-glyceraldehyde-3-phosphate 
      dehydrogenase from Bacillus stearothermophilus.
PG  - 7556-64
AB  - Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from 
      Bacillus stearothermophilus can be described as a dimer of dimers with three 
      nonequivalent interfaces. To investigate the contribution of intra- and 
      intersubunit interactions to GAPDH thermostability, 10 residues located either at 
      the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain 
      (amino acids 149-312) were mutated and the thermal unfolding of the mutants was 
      studied by differential scanning calorimetry in the absence and presence of 
      saturating concentrations of NAD. Disruptions of intrasubunit interactions lead 
      to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. 
      Moreover, for the N313T mutant, a weakening of cooperative interactions between 
      the catalytic and the cofactor domains and an inefficient binding of NAD are 
      observed. This is likely the consequences of modification or loss of the hydrogen 
      bonding network associating N313 and residues 236-238 and N313 and the 
      nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, 
      which are involved in stacking hydrophobic interactions, mutating both positions 
      does not affect the efficiency of NAD binding. This shows that the factors 
      involved in the thermostability of the tetrameric apo GAPDH are then different 
      from those induced by NAD binding. Disruption of intersubunit hydrogen bonds 
      between the catalytic domain and the NAD-binding domain of a neighboring subunit 
      also leads to a significant destabilization of the apo tetrameric form as 
      observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. 
      Both results are likely the consequence of a loss of hydrogen bonds across the 
      P-axis and the Q-axis between D282 and R197 and between D282 and R52, 
      respectively. Similar results, i.e., a destabilizing effect and inefficient NAD 
      binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is 
      introduced at the S-loop of the R-axis-related subunit via mutations at the 
      adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial 
      heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer 
      shows two peaks. Taking into account the recent small-angle X-ray scattering 
      studies which suggested that the dimeric form of the D282G mutant and of the 
      dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, 
      unpublished results), we propose that the presence of one or two peaks in thermal 
      unfolding of dimers is a signature of the dimer type.
FAU - Roitel, Olivier
AU  - Roitel O
AD  - Maturation des ARN et Enzymologie Moleculaire, UMR 7567 CNRS-UHP, Universite 
      Henri Poincare Nancy I, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.
FAU - Ivinova, Olga
AU  - Ivinova O
FAU - Muronetz, Vladimir
AU  - Muronetz V
FAU - Nagradova, Natalia
AU  - Nagradova N
FAU - Branlant, Guy
AU  - Branlant G
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Apoenzymes)
RN  - 0 (Holoenzymes)
RN  - 0 (Peptide Fragments)
RN  - 0 (Recombinant Proteins)
RN  - EC 1.2.1.12 (Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating))
SB  - IM
MH  - Apoenzymes/chemistry/genetics
MH  - Catalytic Domain/genetics
MH  - Dimerization
MH  - Enzyme Stability/genetics
MH  - Geobacillus stearothermophilus/*enzymology/genetics
MH  - Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/*chemistry/genetics
MH  - Holoenzymes/chemistry/genetics
MH  - *Hot Temperature
MH  - Mutagenesis, Site-Directed
MH  - Peptide Fragments/chemistry/genetics
MH  - Protein Denaturation/genetics
MH  - *Protein Folding
MH  - Recombinant Proteins/chemistry
EDAT- 2002/06/12 10:00
MHDA- 2002/07/27 10:01
CRDT- 2002/06/12 10:00
PHST- 2002/06/12 10:00 [pubmed]
PHST- 2002/07/27 10:01 [medline]
PHST- 2002/06/12 10:00 [entrez]
AID - bi012084+ [pii]
AID - 10.1021/bi012084+ [doi]
PST - ppublish
SO  - Biochemistry. 2002 Jun 18;41(24):7556-64. doi: 10.1021/bi012084+.