Cytochrome P450-catalyzed dealkylation of atrazine byRhodococcussp. strain NI86/21 involves hydrogen atom transfer rather than single electron transfer

Abstract

Cytochrome P450 enzymes are responsible for a multitude of natural transformation reactions. For oxidative N-dealkylation, single electron (SET) and hydrogen atom abstraction (HAT) have been debated as underlying mechanisms. Combined evidence from (i) product distribution and (ii) isotope effects indicate that HAT, rather than SET, initiates N-dealkylation of atrazine to desethyl- and desisopropylatrazine by the microorganism Rhodococcus sp. strain NI86/21. (i) Product analysis revealed a non-selective oxidation at both the αC and βC-atom of the alkyl chain, which is expected for a radical reaction, but not SET. (ii) Normal (13)C and (15)N as well as pronounced (2)H isotope effects (εcarbon: -4.0‰ ± 0.2‰; εnitrogen: -1.4‰ ± 0.3‰, KIEH: 3.6 ± 0.8) agree qualitatively with calculated values for HAT, whereas inverse (13)C and (15)N isotope effects are predicted for SET. Analogous results are observed with the Fe(iv)[double bond, length as m-dash]O model system [5,10,15,20-tetrakis(pentafluorophenyl)porphyrin-iron(iii)-chloride + NaIO4], but not with permanganate. These results emphasize the relevance of the HAT mechanism for N-dealkylation by P450.

Deuterium‐ and Tritium‐Labelled Compounds: Applications in the Life Sciences

“…[47] Significant KIEs have been observed for several P450-catalyzed reactions (Scheme 5), [48] particularly for dealkylation reactions of ethers and amides,w hereas amine Ndealkylation and aromatic hydroxylation reactions typically have low KIEs for mechanistic reasons. [49] Aside from P450, other human metabolic enzymes,s uch as the aldehyde oxidase (AO) [50] and the monoamine oxidase (MAO) family, [51] also produce measurable KIEs.This effect is utilized for the quantification of native MAOe nzyme activities,f or example,inrelation to smoking,byadual tracer approach to compare the binding rates of selective PET radiotracers in the presence and absence of an additional deuterium label. [52] Depending on the enzymes involved, and hence the mechanism of the rate-determining step,a nother consequence can be ad ifferent metabolic turnover of ad rug molecule when replacing hydrogen for deuterium.…”

Deuterium‐ und tritiummarkierte Verbindungen: Anwendungen in den modernen Biowissenschaften

“…[46] Die Hauptstoffwechselenzyme im Menschen stammen aus der Cytochrom-P450(CYP)-Enzymfamilie.S ie sind für7 5% des gesamten Wirkstoffmetabolismus verantwortlich und finden daher große Aufmerksamkeit in der pharmazeutischen Industrie. [49] Neben CYPs produzieren auch andere humane Stoffwechselenzyme,w ie beispielsweise die Aldehydoxidase (AO) [50] und die Monoaminoxidasen (MAOs), [51] messbare KIEs.Dieser Effekt wird zur Quantifizierung der Aktivitätnativer MAO-Enzyme (z. [49] Neben CYPs produzieren auch andere humane Stoffwechselenzyme,w ie beispielsweise die Aldehydoxidase (AO) [50] und die Monoaminoxidasen (MAOs), [51] messbare KIEs.Dieser Effekt wird zur Quantifizierung der Aktivitätnativer MAO-Enzyme (z.…”

Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2

“…Two putative mechanistic options have been adopted for the N-methyl hydroxylation, i.e., hydrogen atom transfer (HAT) [25,26] and single-electron transfer (SET) [27]. Originating from the high-spin (HS) quartet and low-spin (LS) doublet states of the active species of CYP1A2 (Compound I, Cpd I in brief), theoretical studies on the initial N-methyl hydroxylation of alkane have explored two possible mechanisms: the two-state reactivity (TSR) scenario [28][29][30] and the spin-selective manner (SSM) [31,32].…”