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 language | English (US) |
|---|---|
| Pages (from-to) | 4785-4798 |
| Number of pages | 14 |
| Journal | Journal of the American Chemical Society |
| Volume | 125 |
| Issue number | 16 |
| DOIs | |
| State | Published - Apr 23 2003 |
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- Chemistry(all)
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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 journal › Article
}
TY - JOUR
T1 - Binding equilibrium isotope effects for glucose at the catalytic domain of human brain hexokinase
AU - Lewis, Brett E.
AU - Schramm, Vern L.
PY - 2003/4/23
Y1 - 2003/4/23
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037462127&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037462127&partnerID=8YFLogxK
U2 - 10.1021/ja0298242
DO - 10.1021/ja0298242
M3 - Article
C2 - 12696897
AN - SCOPUS:0037462127
VL - 125
SP - 4785
EP - 4798
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 16
ER -