Published on Web 04/01/2003
Binding Equilibrium Isotope Effects for Glucose at the
Catalytic Domain of Human Brain Hexokinase
Brett E. Lewis and Vern L. Schramm*
Contribution from The Albert Einstein College of Medicine, 1300 Morris Park AVenue,
Bronx, New York 10461
Received December 19, 2002; E-mail: vern@aecom.yu.edu
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.
Introduction
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