THE CYP2A3-GENE PRODUCT CATALYZES COUMARIN 7-HYDROXYLATION IN HUMAN LIVER-MICROSOMES

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a βgt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160 → His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a λmax at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min−1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 μM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 μM). These data establish that the CYP2A3 gene product is primarily responsible for coumarin 7-hydroxylase activity in human liver. The level of expression of this activity varied up to 40-fold between livers. Levels of IIA mRNA also varied significantly between liver specimens, and three specimens had no detectable mRNA. © 1990, American Chemical Society. All rights reserved.