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 immobilized 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.

Figure 1

a) CYP2C9 with the access channel and reductase binding sites, cysteine residues (yellow), and lysines (gray) with lysine nitrogens (blue). b) Cartoon of the attachment of CYP2C9 to a gold surface coated with OT and MUA (3:1) catalyzed by EDC/NHS. c) Corresponding AFM images prior to enzyme attachement (top) and after attachment (bottom).

Figure 2

Magnetization data as a function of external magnetic field. Curves correspond to CYP2C9 (black), CYP2C9 and dapsone (green), CYP2C9 and flurbiprofen (red), and CYP2C9 and flurbiprofen/dapsone (blue). Data acquired at a temperature of 300 K in increasing and decreasing fields. The CYP2C9 data at high fields was slightly different for the increasing and decreasing field measurements due to a slight change in the sample position during the last part of the experiment.