Human Cytochrome P450 2A7: Stability and Activity Relative to other Human CYP2A Enzymes

Abstract

Humans have three cytochrome P450 (CYP) 2A genes: CYP2A6, CYP2A7, and CYP2A13. CYP2A6 and CYP2A13 proteins metabolize coumarin and other compounds. Although CYP2A7 is 94% identical to CYP2A6, it has been reported that CYP2A7 is an inactive protein. CYP2A7 expressed in vaccinia virus did not yield functional holoprotein as evaluated by spectral analysis, Western blot, and catalytic activity, likely failing to incorporate heme1. Western blot revealed that CYP2A7 protein was produced in COS cells, but this protein did not metabolize coumarin to 7‐hydroxycoumarin2. Our goal is to reevaluate if CYP2A7 is a stable holoprotein capable of metabolizing coumarin or other CYP2A substrates. We have expressed truncated, His‐tagged CYP2A7 in E. coli. This system produces CYP2A7 protein that does incorporate heme, as indicated by a spectral shift in the reduced carbon monoxide difference assay. Generation of purified CYP2A7 protein is enabling new comparisons of human CYP2A stability, ligands, and catalytic activity. Additionally, comparison of the CYP2A7 sequence with the CYP2A6 and CYP2A13 structures suggests substitutions that may be responsible for differences among the human CYP2A enzymes. Research supported by NIH GM76343 (EES).1Yamano, S.; Tatsuno, J.; Gonzalez, F. J. Biochemistry 1990, 29, 1322‐1329.2Ding, S.; Lake, B. G.; Friedberg, T.; Wolf, C. R. Biochem J 1995, 306, 161‐166.