Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.
A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.
Vignais
M L
ML
Laboratoire d'Enzymologie et de Génie Génétique, URA CNRS 457, Vandoeuvre-lès-Nancy, France.
Corbier
C
C
Mulliert
G
G
Branlant
C
C
Branlant
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases