Probing the Functional Role of Phenylalanine-31 of Escherichia coli Dihydrofolate Reductase by Site-Directed Mutagenesis

Jin Tann Chen, Kazunari Taira, Stephen J. Benkovic, Chen Pei D. Tu

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val. The kinetic behavior of the mutant enzymes in general is similar to that of the wild type. The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E•NADPH.H4F ternary complex as determined for the wild type. The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex. The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.

Original languageEnglish (US)
Pages (from-to)4093-4100
Number of pages8
JournalBiochemistry
Volume26
Issue number13
DOIs
StatePublished - 1987

All Science Journal Classification (ASJC) codes

  • Biochemistry

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