Structure of dihydrofolate when bound to dihydrofolate reductase

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31 Scopus citations

Abstract

The Raman spectrum of dihydrofolate (H2folate) complexed with dihydrofolate reductase (DHFR) and NADP+, believed to be an accurate mimic of the productive DHFR/NADPH/H2folate complex involved in the reaction catalyzed by DHFR, contains bands associated with stretch motions of N5=C6 of bound substrate. However, the assignments of these bands, which are of considerable importance to understanding enzymic mechanism and substrate binding, are in doubt. The vibrational spectra of dihydrofolate, alone and complexed with water and with acetate, have been calculated using quantum mechanical ab initio procedures in order to assign the observed bands. Several structural conclusions follow from these calculations. N5 of H2folate when bound to DHFR/NADP+ has a pK(a) of 6.5. From an examination of deuteration shifts, the immediate environment of N5 of substrate is quite hydrophobic; there does not appears to be an immediate water molecule near enough to form a hydrogen bond with a protonated N5-H. It is suggested that the carboxyl group of Asp27, the only ionizable group in the DHFR binding site, is ionized at physiological pH values and does not donate a proton to substrate during enzymic catalysis. Overall, the results suggest that a major structural attribute of DHFR is to raise the pK(a) of N5 4 units when H2folate binds in the productive ground-state ternary complex. Such a strategy would appear to be responsible for a substantial portion of the rate enhancement in the reaction catalyzed by DHFR.

Original languageEnglish (US)
Pages (from-to)7730-7737
Number of pages8
JournalJournal of the American Chemical Society
Volume120
Issue number31
DOIs
StatePublished - Aug 12 1998

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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