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Papers In Press, published online ahead of print February 20, 2001
Department of Medicine Molecular Biology & Pharmacology, Washington University, St. Louis, MO 63110
Corresponding Author: ggrant{at}pcg.wustl.edu
D-3-Phosphoglycerate Dehydrogenase from E. coli contains two Gly-Gly sequences that occur at junctions between domains. A previous study (7) determined that the Gly-Gly sequence at the junction between the regulatory and substrate binding domain functions as a hinge between the domains. Mutations in this area significantly decrease the ability of serine to inhibit activity but have little effect on the Km and kcat . Conversely, the present study shows that mutations to the Gly-Gly sequence at the junction of the substrate and nucleotide binding domain, which form the active site cleft, have a significant effect on the kcat of the enzyme without substantially altering the enzyme?s sensitivity to serine. In addition, mutation of Gly-294, but not Gly-295, has a profound effect on the cooperativity of serine inhibition. Interestingly, even though cooperativity of inhibition can be significantly reduced, there is little apparent effect on the cooperativity of serine binding itself. An additional mutant, G336V, G337V also reduces the cooperativity of inhibition, but in this case, serine binding is also reduced to the point that it cannot be measured by equilibrium dialysis. The double mutant, G294V G336V, demonstrates that strain imposed by mutation at one hinge can be partially relieved by mutation at the other hinge, demonstrating linkage between the two hinge regions. These data show that the two cooperative processes, serine binding and catalytic inhibition, can be uncoupled. Consideration of the allowable torsional angles for the side chains introduced by the mutations yields a range of values for these angles that the glycine residues likely occupy in the native enzyme. Comparison of these values to the torsional angles found for the inhibited enzyme from crystal coordinates provides potential beginning and ending orientations for the transition from active to inhibited enzyme which will allow modeling of the dynamics of domain movement.
J. Biol. Chem, 10.1074/jbc.M009957200
Submitted on November 1, 2000
Revised on February 20, 2001
Accepted on February 20, 2001
Amino acid residue mutations uncouple cooperative effects in E. coli D-3-phosphoglycerate dehydrogenaseÝ
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