scholarly journals Polymorphisms of CYP2C8 Alter First-Electron Transfer Kinetics and Increase Catalytic Uncoupling

2019 ◽  
Vol 20 (18) ◽  
pp. 4626
Author(s):  
William R. Arnold ◽  
Susan Zelasko ◽  
Daryl D. Meling ◽  
Kimberly Sam ◽  
Aditi Das
Keyword(s):  
Hydrogen Peroxide ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Active Site ◽  
Stopped Flow ◽  
Cytochrome P450 Reductase ◽  
Flow Measurements ◽  
Electron Transfer Kinetics ◽  
Naturally Occurring ◽  
Cytochrome P450 2C8

Cytochrome P450 2C8 (CYP2C8) epoxygenase is responsible for the metabolism of over 60 clinically relevant drugs, notably the anticancer drug Taxol (paclitaxel, PAC). Specifically, there are naturally occurring polymorphisms, CYP2C8*2 and CYP2C8*3, that display altered PAC hydroxylation rates despite these mutations not being located in the active site. Herein, we demonstrate that these polymorphisms result in a greater uncoupling of PAC metabolism by increasing the amount of hydrogen peroxide formed per PAC turnover. Anaerobic stopped-flow measurements determined that these polymorphisms have altered first electron transfer kinetics, compared to CYP2C8*1 (wildtype), that suggest electron transfer from cytochrome P450 reductase (CPR) is disfavored. Therefore, these data demonstrate that these polymorphisms affect the catalytic cycle of CYP2C8 and suggest that redox interactions with CPR are disrupted.

scholarly journals Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

2002 ◽  
Vol 367 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Kirsty KNIGHT ◽  
Nigel S. SCRUTTON
Keyword(s):  
Nitric Oxide ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Stopped Flow ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase ◽  
Oxide Synthase ◽  
Reductase Domain

The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+. The second and third steps represent further reduction of the enzyme flavins and NADP+ release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP+ release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+ is modestly slower in calmodulin-depleted enzyme. Our studies indicate the need for significant re-interpretation of published kinetic data for electron transfer in the reductase domain of neuronal nitric oxide synthase.

Differential Contribution of Active Site Residues in Substrate Recognition Sites 1 and 5 to Cytochrome P450 2C8 Substrate Selectivity and Regioselectivity†

Biochemistry ◽  
2004 ◽  
Vol 43 (24) ◽  
pp. 7834-7842 ◽  
Author(s):  
Oranun Kerdpin ◽  
David J. Elliot ◽  
Sanford L. Boye ◽  
Donald J. Birkett ◽  
Krongtong Yoovathaworn ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
Substrate Selectivity ◽  
Active Site Residues ◽  
Recognition Sites ◽  
Cytochrome P450 2C8 ◽  
Differential Contribution

scholarly journals Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison’s disease

2005 ◽  
Vol 152 (1) ◽  
pp. 95-101 ◽  
Author(s):  
L Nikfarjam ◽  
S Kominami ◽  
T Yamazaki ◽  
S Chen ◽  
R Hewer ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Conformational Changes ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Cytochrome P450 Reductase ◽  
Difference Spectra ◽  
Native Form ◽  
Mechanism Of Inhibition

Objective: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. Design: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. Methods: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. Results: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450 nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. Conclusions: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

scholarly journals Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

2020 ◽  
Vol 21 (16) ◽  
pp. 5734
Author(s):  
Joaquin Ramirez-Ramirez ◽  
Javier Martin-Diaz ◽  
Nina Pastor ◽  
Miguel Alcalde ◽  
Marcela Ayala
Keyword(s):  
Hydrogen Peroxide ◽  
Cytochrome P450 ◽  
Active Site ◽  
Bulk Solution ◽  
Computational Studies ◽  
Organic Substrates ◽  
Oxidation Reactions ◽  
Heme Group ◽  
Wide Range

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

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