TY - JOUR
T1 - Measurement of the intramolecular isotope effect on aliphatic hydroxylation by chromobacterium violaceum phenylalanine hydroxylase
AU - Panay, Aram J.
AU - Fitzpatrick, Paul F.
PY - 2010/4/28
Y1 - 2010/4/28
N2 - The non-heme iron enzyme phenylalanine hydroxylase from "genus-species" Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined in order to gain insight into the reactivity of the iron center. With 3-[2H 11-cyclohexyl]alanine as the substrate, the isotope effect on the kcat value was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently, the isotope effect was determined as an intramolecular effect by measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6- 2H6-cyclohexyl]alanine. The ratio of 4-HO- cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This gave a calculated value of 12.6 for the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum-mechanical tunneling to the reaction.
AB - The non-heme iron enzyme phenylalanine hydroxylase from "genus-species" Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined in order to gain insight into the reactivity of the iron center. With 3-[2H 11-cyclohexyl]alanine as the substrate, the isotope effect on the kcat value was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently, the isotope effect was determined as an intramolecular effect by measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6- 2H6-cyclohexyl]alanine. The ratio of 4-HO- cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This gave a calculated value of 12.6 for the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum-mechanical tunneling to the reaction.
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U2 - 10.1021/ja101563t
DO - 10.1021/ja101563t
M3 - Article
C2 - 20355730
AN - SCOPUS:77952557540
VL - 132
SP - 5584
EP - 5585
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 16
ER -