Site‐specific mutagenesis of dihydrofolate reductase from Escherichia coli

Jin‐Tann ‐T Chen, Ruth J. Mayer, Carol A. Fierke, Stephen J. Benkovic

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Two site‐specific mutations of dihydrofolate reductase from Escherichia coli based on the x‐ray crystallographic structure were constructed. The first mutation (His‐45 → Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH. The second (Thr‐113 → Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5‐;C6 imine undergoing reduction. The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6–40‐fold. A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild‐type enzyme. The second was demonstrated to increase the dissociation rate constant for methotrexate 20–30‐fold, and presumably dihydrofolate also, with it corresponding 20–30‐fold increase in the dissociation constant. In this case an identical effect was noted on V/K but not in V relative to the native enzyme. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.

Original languageEnglish (US)
Pages (from-to)73-82
Number of pages10
JournalJournal of cellular biochemistry
Volume29
Issue number2
DOIs
StatePublished - 1985

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Mutagenesis
Tetrahydrofolate Dehydrogenase
Escherichia coli
Mutation
Enzymes
Rate constants
Imines
Hydrogen Bonding
NADP
Methotrexate
Protons
X-Rays
Hydrogen bonds
Association reactions
Substrates
dihydrofolate

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Chen, Jin‐Tann ‐T ; Mayer, Ruth J. ; Fierke, Carol A. ; Benkovic, Stephen J. / Site‐specific mutagenesis of dihydrofolate reductase from Escherichia coli. In: Journal of cellular biochemistry. 1985 ; Vol. 29, No. 2. pp. 73-82.
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Site‐specific mutagenesis of dihydrofolate reductase from Escherichia coli. / Chen, Jin‐Tann ‐T; Mayer, Ruth J.; Fierke, Carol A.; Benkovic, Stephen J.

In: Journal of cellular biochemistry, Vol. 29, No. 2, 1985, p. 73-82.

Research output: Contribution to journalArticle

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