PMID- 15606760
OWN - NLM
STAT- MEDLINE
DCOM- 20050706
LR  - 20161017
IS  - 0014-2956 (Print)
IS  - 0014-2956 (Linking)
VI  - 271
IP  - 23-24
DP  - 2004 Dec
TI  - Involvement of two positively charged residues of Chlamydomonas reinhardtii 
      glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme 
      complex involved in CO2 assimilation.
PG  - 4737-44
AB  - The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of 
      Chlamydomonas reinhardtii is part of a complex that also includes 
      phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved 
      in protein-protein interactions in this complex, by changing residues K128 and 
      R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the 
      wild type and a lower catalytic constant, whatever the cofactor. The kinetics of 
      the mutant R197A were similar to those of the wild type, while the R197E mutant 
      had a lower catalytic constant with NADPH. Only small structural changes near the 
      mutation may have caused these differences, since circular dichroism and 
      fluorescence spectra were similar to those of wild-type GAPDH. Molecular 
      modelling of the mutants led to the same conclusion. All mutants, except R197E, 
      reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants 
      measured by surface plasmon resonance were 10-70-fold higher with the mutants 
      than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we 
      calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of 
      the stabilizing effect of a salt bridge for the interaction between GAPDH and 
      CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to 
      form the native complex. The absence of kinetic changes of all the mutant 
      GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants 
      do not undergo the conformation change essential for PRK binding.
FAU - Graciet, Emmanuelle
AU  - Graciet E
AD  - Laboratoire Genetique et Membranes, Departement Biologie Cellulaire, Institut 
      Jacques Monod, UMR 7592 CNRS, Universites Paris VI-VII, Paris, France.
FAU - Mulliert, Guillermo
AU  - Mulliert G
FAU - Lebreton, Sandrine
AU  - Lebreton S
FAU - Gontero, Brigitte
AU  - Gontero B
LA  - eng
PT  - Journal Article
PL  - England
TA  - Eur J Biochem
JT  - European journal of biochemistry
JID - 0107600
RN  - 142M471B3J (Carbon Dioxide)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
RN  - EC 2.7.1.- (Phosphotransferases (Alcohol Group Acceptor))
RN  - EC 2.7.1.19 (phosphoribulokinase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Carbon Dioxide/*metabolism
MH  - Catalysis
MH  - Chlamydomonas reinhardtii/*enzymology
MH  - Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry/genetics/*metabolism
MH  - Kinetics
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism
MH  - Sequence Homology, Amino Acid
EDAT- 2004/12/21 09:00
MHDA- 2005/07/07 09:00
CRDT- 2004/12/21 09:00
PHST- 2004/12/21 09:00 [pubmed]
PHST- 2005/07/07 09:00 [medline]
PHST- 2004/12/21 09:00 [entrez]
AID - EJB4437 [pii]
AID - 10.1111/j.1432-1033.2004.04437.x [doi]
PST - ppublish
SO  - Eur J Biochem. 2004 Dec;271(23-24):4737-44. doi: 
      10.1111/j.1432-1033.2004.04437.x.