Binding equilibrium isotope effects for glucose at the catalytic domain of human brain hexokinase

Brett E. Lewis, Vern L. Schramm

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

We have utilized tritium isotope effects to probe the in vitro binding equilibrium between glucose and human brain hexokinase (E.C.2.7.1.1). Replacing a backbone hydrogen atom in glucose with tritium can significantly increase or decrease the equilibrium association constant. Specifically, the equilibrium tritium isotope effects are 1.027 ± 0.002, 0.927 ± 0.0003, 1.027 ± 0.004, 1.051 ± 0.001, 0.988 ± 0.001, and 1.065 ± 0.003 for [1-t]-, [2-t]-, [3-t]-, [4-t]-, [5-t]-, and [6,6-t2]glucose, respectively. We have shown that the existence of prebinding equilibrium isotope effects can contribute to binding isotope effect studies but that this effect is insignificant for glucose binding to hexokinase. The binding isotope effects are interpreted in the context of structural studies of hexokinase - glucose complexes. Ab initio calculations on 2-propanol with or without a hydrogen bonding partner, in steric collision with formaldehyde or methane, and on ethanol, cyclohexanol and 1-hydroxymethyl-tetrahydropyran are presented to clarify the magnitude of isotope effects possible in such interactions and the accompanying changes in free energy. Position-specific binding isotope effects provide direct evidence of the partial deprotonation and activation of O6 by Asp657, of other hydrogen bonding interactions with ionic residues, and of the steric compression of CH2 by the backbone carbonyl of Ser603.

Original languageEnglish (US)
Pages (from-to)4785-4798
Number of pages14
JournalJournal of the American Chemical Society
Volume125
Issue number16
DOIs
StatePublished - Apr 23 2003

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Hexokinase
Isotopes
Glucose
Brain
Catalytic Domain
Tritium
Hydrogen Bonding
Hydrogen bonds
Cyclohexanols
Deprotonation
2-Propanol
Methane
Propanol
Formaldehyde
Free energy
Hydrogen
Ethanol
Chemical activation
Association reactions
Atoms

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Binding equilibrium isotope effects for glucose at the catalytic domain of human brain hexokinase. / Lewis, Brett E.; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 125, No. 16, 23.04.2003, p. 4785-4798.

Research output: Contribution to journalArticle

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