Effects of Distal Point-Site Mutations on the Binding and Catalysis of Dihydrofolate Reductase from Escherichia coli

Joseph Adams, Kenneth Johnson, Robert Matthews, Stephen Benkovic

Research output: Contribution to journalArticle

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Abstract

The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 Å away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to >45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 μM for H45Q (similar to wild type) but from 8 to 5000 μM for R44L.

Original languageEnglish (US)
Pages (from-to)6611-6618
Number of pages8
JournalBiochemistry
Volume28
Issue number16
DOIs
StatePublished - Aug 1 1989

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Tetrahydrofolate Dehydrogenase
Catalysis
NADP
Point Mutation
Escherichia coli
Mutagenesis
Mutation
Coenzymes
Site-Directed Mutagenesis
Binding energy
Hydrides
Conformations
Salts
Fluorescence
Experiments
Phosphates
Binding Sites
Enzymes
5,6,7,8-tetrahydrofolic acid

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Adams, Joseph ; Johnson, Kenneth ; Matthews, Robert ; Benkovic, Stephen. / Effects of Distal Point-Site Mutations on the Binding and Catalysis of Dihydrofolate Reductase from Escherichia coli. In: Biochemistry. 1989 ; Vol. 28, No. 16. pp. 6611-6618.
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abstract = "The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 {\AA} away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to >45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 μM for H45Q (similar to wild type) but from 8 to 5000 μM for R44L.",
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Effects of Distal Point-Site Mutations on the Binding and Catalysis of Dihydrofolate Reductase from Escherichia coli. / Adams, Joseph; Johnson, Kenneth; Matthews, Robert; Benkovic, Stephen.

In: Biochemistry, Vol. 28, No. 16, 01.08.1989, p. 6611-6618.

Research output: Contribution to journalArticle

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T1 - Effects of Distal Point-Site Mutations on the Binding and Catalysis of Dihydrofolate Reductase from Escherichia coli

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AU - Johnson, Kenneth

AU - Matthews, Robert

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N2 - The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 Å away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to >45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 μM for H45Q (similar to wild type) but from 8 to 5000 μM for R44L.

AB - The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 Å away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to >45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 μM for H45Q (similar to wild type) but from 8 to 5000 μM for R44L.

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