12/3/2023 0 Comments Cytochromes p450 a success story![]() Ĭryle MJ, De Voss JJ (2008) The role of the conserved threonine in P450 BM3 oxygen activation: substrate-determined hydroxylation activity of the Thr268Ala mutant. Ĭoon MJ (2005) CYTOCHROME P450: nature’s most versatile biological catalyst. J Pharm Sci 11(4):68–82Ĭhilds BRE, Bardsley WG (1975) The steady-state kinetics of peroxidase with 2,2′-azino-di-(3-ethylbenzthiazoline- 6-sulphonic acid) as chromogen. Çelik H, Arinç E (2008) Bioreduction of idarubicin and formation of ROS responsible for DNA cleavage by NADPH-cytochrome P450 reductase and its potential role in the antitumor effect. Ĭarmichael AB, Wong LL (2001) Protein engineering of Bacillus megaterium CYP102: the oxidation of polycyclic aromatic hydrocarbons. Ĭabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. īornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. īlanck J, Ristau O, Zhukov AA, Archakov AI, Rein H, Ruckpaul K (1991) Cytochrome P-450 spin state and leakiness of the monooxygenase pathway. īernhardt R (2006) Cytochromes P450 as versatile biocatalysts. We demonstrate the applicability of the assay, which provides a versatile basis for a high-throughput preliminary screening of P450 enzyme libraries without the need for GC or HPLC analysis and clear indication of the extent of hydrogen peroxide uncoupling.Ītkins WM, Sligar SG (1988) Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam: kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate. This was exemplified for the P450 monooxygenase from Bacillus megaterium (P450 BM3) and five mutants, using different substrates. To measure this uncoupling, we herein report a microtiter plate-based assay for the simultaneous quantification of hydrogen peroxide formation and NAD(P)H consumption using Ampliflu™ Red as reporter. Hence, measuring NAD(P)H consumption only can lead to an overestimation of substrate conversion. This so-called uncoupling of the reaction cycle drains electrons from the system, which consequently does not lead to the desired product, but merely H 2O 2 formation with stoichiometric consumption of NAD(P)H. ![]() These intermediates can decay easily, releasing the reactive oxygen species superoxide anion and hydrogen peroxide (H 2O 2), which can also be further reduced to water. The thereby generated iron-oxygen complex undergoes multiple reductive steps forming different activated oxygen species. Through the electron transfer from NAD(P)H to the heme-center of the P450, the enzyme becomes activated and binds oxygen. In contrast to most other enzymes, the activity of P450 enzymes is not only dependent on substrate and cofactor availability and reaction conditions, but also depends on the coupling efficiency of the catalytic cycle itself. These monooxygenases catalyze the incorporation of a single atom of molecular oxygen into their substrate. ![]() Cytochrome P450s belong to a large and diverse group of heme-containing enzymes.
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