Measurement of the Intramolecular Isotope Effect on Aliphatic Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase

Abstract

The non-heme iron enzyme phenylalanine hydroxylase from 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 to gain insight into the reactivity of the iron center. With 3-[ 2 H 11 -cyclohexyl]-alanine as substrate, the isotope effect on the k cat 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, measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6-2 H 6 -cyclohexyl]-alanine. The ratio of 4-HO-cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This allowed calculation of 12.6 as 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.Phenylalanine hydroxylase (PheH) is a non-heme iron dependent monooxygenase that catalyzes the hydroxylation of the amino acid phenylalanine to yield tyrosine (Scheme 1) 1 . PheH is found in organisms ranging from bacteria to humans. In mammals, the enzyme is responsible for catabolism of dietary phenylalanine, and mutations in PheH are linked to the disorder phenylketonuria 2 . Among the bacterial enzymes, that from Chromobacterium violaceum (CvPheH) is the most studied3 -6. PheH is a member of the family of aromatic amino acid hydroxylases, along with tyrosine hydroxylase (TyrH) and tryptophan

Evidence for a High-Spin Fe(IV) Species in the Catalytic Cycle of a Bacterial Phenylalanine Hydroxylase

“…However, CvPheH will also catalyze the hydroxylation of cyclohexylalanine. This aliphatic hydroxylation reaction does exhibit a large deuterium kinetic isotope effect (∼15) (19), and amino acid hydroxylation is reported to be fully coupled to tetrahydropterin oxidation for this substrate (33). Similar rapid-quench Mössbauer experiments were carried out with deuterated cyclohexylalanine in an attempt to increase the amount of the Fe(IV) intermediate, but the intermediate could not be detected with this substrate.…”

“…In the case of the bacterial PheH, the structure of the ferric enzyme shows a bidentate glutamate , in contrast to the eukaryotic enzymes. Still, the reactivities of the hydroxylating intermediates in the eukaryotic and prokaryotic enzymes appear to be essentially identical on the basis of their similar abilities to catalyze aromatic, benzylic, and aliphatic hydroxylation − .…”

Phenylalanine meta‐Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation

“…Aromatic amino acid hydroxylases have been demonstrated to operate by a mechanism that involves an NIH shift. In all previous mechanistic studies (typically with Trp and Phe hydroxylases) the deuterium or tritium label from the hydroxylation position was present in >60 % of the product . Unexpectedly, in this work with Phe3H, the second deuterium atom was lost.…”

“…Isotopic labelling has been used extensively to study the catalytic mechanisms of aromatic amino acid hydroxylases . In this work, substrate deuterium labelling was employed to probe the mechanism of Phe3H .…”

Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations

“…With the intent to augment its accumulation, we used the perdeuterated cyclohexylalanine analog. Despite the fact that previous studies have shown that the reaction of Cv PheH with this analog results in hydroxylation of one of its aliphatic C-H bonds, exhibits a deuterium kinetic isotope effect of ∼15, and is fully coupled [75], the ferryl intermediate does not accumulate to a detectable level, as judged by the absence of the well-resolved high-energy line of the ferryl complex in the Mössbauer spectra, in the reaction of Cv PheH with the perdeuterated analog, presumably because addition of O 2 (and therefore formation of the intermediate) is significantly slowed by the diminished ST efficacy of the non-native substrate [73]. …”