Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148.
A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.
Corbier
C
C
Laboratoire d'Enzymologie et de Génie Génétique--UA CNRS 457, Faculté des Sciences, Vandoeuvre les Nancy, France.
Branlant
C
C
Wonacott
A
A
Branlant
G
G
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D011487:Protein Conformation; D012694:Serine / Q000502:physiology*; D013237:Stereoisomerism; D013379:Substrate Specificity
D012694:Serine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases