A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy

J. Zheng, Y. Q. Chen, Robert Callender

Research output: Contribution to journalArticle

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Abstract

We report here the Raman spectra of NADPH, NADP+, 3-acetylpyridine adenine dinucleotide (AcPdADP+), NADH and a fragment of these molecules, 2'-phospho-adenosine-5'-diphosphoribose (Ado2'p5'ppRib), bound to Escherichia coli dihydrofolate reductase (DHFR). The positions that are observed for the bound adenosine 'triplet' bands are consistent with a protein binding pocket for this group which is quite hydrophobic in nature. No binding effect is observed on Raman bands associated with the nicotinamide group of NADP+ as a binary complex with DHFR, suggesting very loose, if any, binding of this group. In contrast, changes in the Raman spectrum of the nicotinamide group of NADP+ bound to an inhibitor (trimethoprim) ternary complex of DHFR are clearly observed which indicate substantial binding interaction. The carboxamide group of bound NADPH (and NADH) adopts the trans conformation. A 35-cm-1 upshift is observed in the rocking motion of the carboxamide -NH2 group of NADPH, and a 5-cm-1 upward shift is seen in the C=O stretch mode of AcPdADP+ upon binding to the enzyme-trimethoprim complex. These results suggest that the -NH2 group of the carboxamide moiety is more tightly hydrogen bonded in the protein binding pocket than in solution while that of the C=O group is less tightly hydrogen bonded; these hydrogen bonds would appear to be responsible for holding the nicotinamide headgroup in place properly for catalysis. We have compared this with the results obtained previously in other protein complexes, and interpret the observed shifts in these bands as a measure of the hydrogen bonding enthalpy of the -NH2 and C = O groups with their protein environments. Perhaps surprisingly, the magnitude of the hydrogen bonding enthalpy takes on a limited number of discrete values over five protein complexes rather than over a continuous range. The effect that this has on the catalytic properties of DHFR and the other NAD dehydrogenases that we have studied to date, particularly their stereochemistry, is discussed. A small downward shift is observed for the P-O stretch of the 2'-phosphate moiety of NADP. This indicates that the 2'-phosphate moiety binds to DHFR in the dianionic form. Furthermore, the local enthalpic interaction that the 2'-phosphate group has with protein is stronger than its interaction with water.

Original languageEnglish (US)
Pages (from-to)9-16
Number of pages8
JournalEuropean Journal of Biochemistry
Volume215
Issue number1
StatePublished - 1993
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Raman Spectrum Analysis
Coenzymes
NADP
Spectroscopy
Niacinamide
NAD
Hydrogen bonds
Trimethoprim
Phosphates
Hydrogen
Hydrogen Bonding
Raman scattering
Enthalpy
Protein Binding
Proteins
Adenosine Diphosphate Ribose
Stereochemistry
Adenosine
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

Cite this

A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy. / Zheng, J.; Chen, Y. Q.; Callender, Robert.

In: European Journal of Biochemistry, Vol. 215, No. 1, 1993, p. 9-16.

Research output: Contribution to journalArticle

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