γ-Butyrobetaine hydroxylase: Stereochemical course of the hydroxylation reaction

Sasha Englard, John S. Blanchard, Christian F. Midelfort

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

The stereochemical course of the aliphatic hydroxylation of γ-butyrobetaine by calf liver and by Pseudomonas sp AK1 γ-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-γ-butyrobetaine or [3(R)-3-3H]-γ-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-γ-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-γ-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-γ-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-γ-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate α-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for γ-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.

Original languageEnglish (US)
Pages (from-to)1110-1116
Number of pages7
JournalBiochemistry
Volume24
Issue number5
StatePublished - 1985

Fingerprint

gamma-Butyrobetaine Dioxygenase
Hydroxylation
Tritium
Liver
Carnitine
Substrates
Mixed Function Oxygenases
Biochemistry
Deuterium
Pseudomonas
gamma-butyrobetaine
Oxidants
Isotopes
Hydrogen

ASJC Scopus subject areas

  • Biochemistry

Cite this

γ-Butyrobetaine hydroxylase : Stereochemical course of the hydroxylation reaction. / Englard, Sasha; Blanchard, John S.; Midelfort, Christian F.

In: Biochemistry, Vol. 24, No. 5, 1985, p. 1110-1116.

Research output: Contribution to journalArticle

Englard, Sasha ; Blanchard, John S. ; Midelfort, Christian F. / γ-Butyrobetaine hydroxylase : Stereochemical course of the hydroxylation reaction. In: Biochemistry. 1985 ; Vol. 24, No. 5. pp. 1110-1116.
@article{41cec8507df24dc0bf1e5ab11cfd3d40,
title = "γ-Butyrobetaine hydroxylase: Stereochemical course of the hydroxylation reaction",
abstract = "The stereochemical course of the aliphatic hydroxylation of γ-butyrobetaine by calf liver and by Pseudomonas sp AK1 γ-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-γ-butyrobetaine or [3(R)-3-3H]-γ-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-γ-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-γ-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-γ-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9{\%}. Experiments using racemic [methyl-14C,3(RS)-3-3H]-γ-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate α-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for γ-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.",
author = "Sasha Englard and Blanchard, {John S.} and Midelfort, {Christian F.}",
year = "1985",
language = "English (US)",
volume = "24",
pages = "1110--1116",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

}

TY - JOUR

T1 - γ-Butyrobetaine hydroxylase

T2 - Stereochemical course of the hydroxylation reaction

AU - Englard, Sasha

AU - Blanchard, John S.

AU - Midelfort, Christian F.

PY - 1985

Y1 - 1985

N2 - The stereochemical course of the aliphatic hydroxylation of γ-butyrobetaine by calf liver and by Pseudomonas sp AK1 γ-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-γ-butyrobetaine or [3(R)-3-3H]-γ-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-γ-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-γ-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-γ-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-γ-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate α-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for γ-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.

AB - The stereochemical course of the aliphatic hydroxylation of γ-butyrobetaine by calf liver and by Pseudomonas sp AK1 γ-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-γ-butyrobetaine or [3(R)-3-3H]-γ-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-γ-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-γ-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-γ-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-γ-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate α-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for γ-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.

UR - http://www.scopus.com/inward/record.url?scp=0021920839&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021920839&partnerID=8YFLogxK

M3 - Article

C2 - 4096892

AN - SCOPUS:0021920839

VL - 24

SP - 1110

EP - 1116

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -