Probing the functional role of threonine-113 of Escherichia coli dihydrofolate reductase for its effect on turnover efficiency, catalysis, and binding.
The role of Thr-113 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Thr-113, a strictly conserved residue that forms a hydrogen bond to the active-site Asp-27 and to the amino group of methotrexate through a fixed water molecule, was replaced by valine. The kinetic scheme is identical in form with the wild-type scheme, although many of the rate constants vary, including a decrease in the association rate constants and an increase in the dissociation rate constants for folate ligands, a decrease in the hydride-transfer rate constant in both directions, and an increase in the intrinsic pKa of Asp-27. Overall, replacement of Thr-113 by Val decreases the binding of folate substrates by approximately 2.3 kcal/mol. These multiple complex changes on various ground and transition states underscore the optimal properties of a strictly conserved residue in the evolution of catalytic function.
Fierke
C A
CA
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710.
Benkovic
S J
SJ
D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009154:Mutation; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D013912:Threonine; D014633:Valine
D013912:Threonine; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D014633:Valine