Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold.
On the basis of the three-dimensional structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of sequence comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, a series of mutants of GAPDH from Bacillus stearothermophilus have been constructed. The results deduced from kinetic and binding studies suggest that the absence of activity of the wild-type GAPDH with NADP as a cofactor is the consequence of at least three factors: (1) steric hindrance, (2) electrostatic repulsion between the charged carboxyl group of Asp32 and the 2'PO4, and (3) structural determinants at the subunit interface of the tetramer. The best value for kcat/KM and KD for NADP was observed for the D32A-L187A-P188S mutant. This triple mutation leads to a switch in favor of NADP specificity but with a kcat/KM ratio 50- and 80-fold less than that observed for the wild type with NAD and for the chloroplast GAPDH with NADP, respectively. Substituting the invariant chloroplastic Thr33-Gly34-Gly35 for the B. stearothermophilus Leu33-Thr34-Asp35 residues on the double mutant Ala187-Ser188 does not improve significantly the affinity for NADP while substituting Ala32 for Asp32 on the double mutant does. Clearly, other subtle adjustments in the adenosine subsite are needed to reconcile the presence of the carboxylate group of Asp32 and the 2'-phosphate of NADP. Kinetic studies indicate a change of the rate-limiting step for the mutants. This could be the consequence of an incomplete apo-holo transition.(ABSTRACT TRUNCATED AT 250 WORDS)
Clermont
S
S
Laboratoire d'Enzymologie et de Génie Génétique, Université de Nancy I, URA CNRS 457, BP 239, Vandoeuvre-lès-Nancy, France.
Corbier
C
C
Mely
Y
Y
Gerard
D
D
Wonacott
A
A
Branlant
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases