pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by raman difference spectroscopy

Yong Qing Chen, Joseph Kraut, Robert Callender

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38 Citations (Scopus)

Abstract

The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR). It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pK(a) of about 6.5. The group responsible for this pK(a) is often assumed to be the carboxyl group of Asp- 27. To determine the ionization state of this carboxyl and its pK(a), we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ. The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the method's limits and problems. The Raman difference spectrum between wild-type ecDHFR and the Asp- 27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken. No protonation of the carboxyl group was detected, implying that its pK(a) is probably less than 5.0. We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pK(a) of 6.3, indicating that the ape enzyme undergoes a pH-dependent conformational change. Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR.

Original languageEnglish (US)
Pages (from-to)936-941
Number of pages6
JournalBiophysical Journal
Volume72
Issue number2 I
StatePublished - Feb 1997

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Tetrahydrofolate Dehydrogenase
Raman Spectrum Analysis
Escherichia coli
Catalytic Domain
Hominidae
Carboxylic Acids
Serine
Proteins
Binding Sites
Ligands
Enzymes

ASJC Scopus subject areas

  • Biophysics

Cite this

pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by raman difference spectroscopy. / Chen, Yong Qing; Kraut, Joseph; Callender, Robert.

In: Biophysical Journal, Vol. 72, No. 2 I, 02.1997, p. 936-941.

Research output: Contribution to journalArticle

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