Probing the Functional Role of Threonine-113 of Escherichia coli Dihydrofolate Reductase for Its Effect on Turnover Efficiency, Catalysis, and Binding

C. A. Fierke, Stephen Benkovic

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Abstract

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 ≈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.

Original languageEnglish (US)
Pages (from-to)478-486
Number of pages9
JournalBiochemistry
Volume28
Issue number2
DOIs
Publication statusPublished - Jan 1 1989

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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