Simultaneous measurement of CYP1A2 activity, regioselectivity, and coupling: Implications for environmental sensitivity of enzyme–substrate binding

Abstract

The cytochrome P450 (CYP) reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature. A high-throughput, optical method was developed to simultaneously measure the rate of substrate depletion, NADPH depletion, and generation of the O-dealkylated product. Increasing the phosphate buffer concentration and temperature increased both the NADPH and EOMCC depletion rates by 6-fold, whereas coupling was constant at 7.9% and the regioselectivity of O-dealkylation to other coupled pathways was constant at 21.7%. Varying the buffer type and pH increased NADPH depletion by 2.5-fold and EOMCC depletion by 3.5-fold; however, neither coupling nor regioselectivity was constant, with variations of 14.4% and 21.6%, respectively. Because the enzyme-substrate binding interaction is a primary determinant of both coupling and regioselectivity, it is reasonable to conclude that ionic strength, as varied by the buffer concentration, and temperature alter the rate without affecting binding while buffer type and pH alter both.

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity

“…Other screening methods have relied on factors that are common to all or a significant proportion of P450dependent reactions. Unfortunately, this assay is expensive to undertake in high throughput because of the cost of the cofactor, and in many P450 systems, the coupling efficiency is poor, making NADPH consumption unrepresentative of product formation [84]. Unfortunately, this assay is expensive to undertake in high throughput because of the cost of the cofactor, and in many P450 systems, the coupling efficiency is poor, making NADPH consumption unrepresentative of product formation [84].…”

Functional Screening of Cytochrome P450 Activity and Uncoupling by Capillary Electrophoresis

“…The catalytic rate for the wild type enzyme was the highest but the coupling efficiency of the wildtype and the mutants was less than 3% (product formed/cofactor consumption x 100%); coupling efficiency for CYP1A2 and a synthetic coumarin based substrate was under 8% (Traylor et al, 2011). Human CYP3A4, bioengineered with a Bacillus megaterium reductase domain showed coupling efficiency of less than 7% for all concentrations of the substrate erythromycin ).…”

“…Table 1). CYP coupling efficiency, estimated based on the mean rate of NADP + to product turn-over ratio (Traylor et al, 2011), ranged from 2-100% (Figure 2). NADP + generation often greatly overestimated actual product formation (Table 1) due to the significant CYP uncoupling in xenobiotic metabolism (Zangar et al, 2004).…”

“…Zebrafish CYP1 activity based on product turnover (fluorescence) relative to rates of NADP + generation (CE) reflected a range of uncoupling among the fluorogenic substrates (Table ). CYP coupling efficiency, estimated based on the mean rate of NADP + to product turnover ratio (i.e., CYP rate by fluorimeter/CYP rate by CE × 100%), , ranged from 2 to 100% (Figure ). NADP + generation often greatly overestimated actual product formation (Table ) due to the significant CYP uncoupling in xenobiotic metabolism .…”

“…CYP1D1 and CYP1B1 had the lowest hydroxylation rates with poor coupling (2.3 and 3.7%, respectively). The excessive reducing equivalents of NADPH not utilized for product formation upon substrate binding was attributed largely to H 2 O 2 release, based on dichlorofluorescein (DCF) as a H 2 O 2 -selective fluorogenic probe (Figure ); H 2 O 2 was generated in uncoupled reactions for human CYP1A2 and CYP1B1 . Measurable H 2 O 2 was present even in well coupled reactions (Figure ).…”