Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: Mechanistic implications

Yong Qing Chen, Robert Callender, Raymond L. Blakley, Robert Callender

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Abstract

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

Original languageEnglish (US)
Pages (from-to)7021-7026
Number of pages6
JournalBiochemistry
Volume33
Issue number23
StatePublished - 1994
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Raman Spectrum Analysis
NADP
Raman spectroscopy
Pteridines
Molecules
Protons
Pterins
Water
Coenzymes
Enzymes
Static Electricity
Serine
Escherichia coli
Hydrogen
Catalytic Domain
Hydrogen bonds
Binding Sites
Electric fields
dihydrofolate

ASJC Scopus subject areas

  • Biochemistry

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Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase : Mechanistic implications. / Chen, Yong Qing; Callender, Robert; Blakley, Raymond L.; Callender, Robert.

In: Biochemistry, Vol. 33, No. 23, 1994, p. 7021-7026.

Research output: Contribution to journalArticle

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