The effect of hinge mutations on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate dehydrogenase.
D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly(336)-Gly(337), is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6 degrees in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn190 to Asn190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the half-the-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.
Dey
Sanghamitra
S
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
Hu
Zhiqin
Z
Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Xu
Xiao Lan
XL
Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Sacchettini
James C
JC
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
Grant
Gregory A
GA
Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; Medicine, Washington University School of Medicine, St. Louis, Missouri 63110. Electronic address: ggrant@wustl.edu.
D001216:Asparagine / Q000737:chemistry; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D050543:Phosphoglycerate Dehydrogenase / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D012694:Serine; D001216:Asparagine; D050543:Phosphoglycerate Dehydrogenase; D014633:Valine; D005998:Glycine