Preparation, Characterization, and Substrate Metabolism of Gold-Immobilized Cytochrome P450 2C9

Abstract

The cytochrome P450 enzymes represent an important class of heme containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological based chips for standardizing cytochrome P450 metabolism or for high throughput screening of pharmaceutical agents. Previous studies have generally immobilize P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed. The cytochrome P450 enzymes represent an important class of heme containing enzymes. They are responsible for a significant portion of xenobiotic metabolism and have been extensively studied for mechanistic and practical reasons. More recently there has been interest in immobilizing these enzymes on a surface to study the electron transfer in a single enzyme, 1 homodimer or heterodimer interactions, 2 or for construction of biological based chips for high throughput screening of pharmaceutical agents.Previous efforts to immobilize P450 enzymes have been made 3 including CYP2E1 on gold electrodes 1 bacterial P450 BM3 on graphite, 4 P450cam immobilized in sol-gel films, 5 pgannett@hsc.wvu.edu. CYP1A2 and CYP3A4 in polyion films, 6 and CYP119 in dimethyldidodecylammonium poly (p-styrene sulfonate). 5 In these studies the detection of substrate binding is often made electrochemically by observing shifts in the redox potential upon substrate binding. 7 Enzymelike metabolic reactions have also been observed by the application of an electrochemical current. 1 However, to be amenable to study and manipulation at the single molecule level it may be desirable to minimize the surrounding matrix. Also, it is highly desirable for the enzyme to metabolize substrates utilizing endogenous enzymes and co-factors to more closely model the corresponding biological system. To our knowledge, this has not been achieved with cytochrome P450 enzymes. NIH Public AccessIn this work we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is dependent upon the linkers used to attach CYP2C9 and to the presence of key substrates during the attachment. A novel method to detect substrate binding, namely superconducti...

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

“…Equation 1 was used to calculate n (0.72) by obtaining the slope of the linearly fitted curve of i p versus v. Furthermore, given n ϭ 0.72, the surface coverage of the CYP2C9 molecules can be calculated (8.0 ϫ 10 Ϫ13 or 4.8 ϫ 10 11 mol/cm 2 ). This number is within a factor of 2 of our previous estimate based on atomic force microscopy (Gannett et al, 2006).…”

“…MUA, 1-octanethiol (OT), N-((3-dimethylamino)-propyl)-N-ethyl carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), flurbiprofen, dapsone, NADPH, and warfarin were purchased from Sigma-Aldrich (St. Louis, MO). CYP2C9 was prepared by expression in an Escherichia coli system, isolated, and purified as described previously (Gannett et al, 2006). Cytochrome P450 reductase was purchased from BD Biosciences (San Jose, CA).…”

“…at ASPET Journals on May 11, 2018 dmd.aspetjournals.org previous estimate based on atomic force microscopy measurement (Gannett et al, 2006). In addition, n can also be used to calculate the electron transfer rate constant (k s ; eq.…”

“…In a previously study (Gannett et al, 2006), we bonded CYP2C9 molecules via the N terminus to the carboxylic acid group of 11-mercaptoundecanoic acid (MUA), resulting in a system with catalytic activity toward substrate. However, this system was still dependent on the presence of NADPH and CPR, both of which are expensive, require continual addition upon extended incubations, and can confound isolation and purification of metabolites.…”

Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism

“…Controlled immobilization of P450s has recently been used to allow better understanding of metabolism (Davydov et al, 2005;Gannett et al, 2006;Mak et al, 2010;Fantuzzi et al, 2011;Panicco et al, 2011;Wollenberg et al, 2012;Bostick et al, 2015) and the formation of homomeric/heteromeric complexes (Backes and Kelley, 2003). We developed a scheme whereby soluble P450 is covalently attached to a self-assembled monolayer (SAM) on a gold film.…”