STRUCTURE-FUNCTION ANALYSIS OF CYP2A10 AND CYP2A11, P450 CYTOCHROMES THAT DIFFER IN ONLY 8 AMINO-ACIDS BUT HAVE STRIKINGLY DIFFERENT ACTIVITIES TOWARD TESTOSTERONE AND COUMARIN

Cytochrome P450 NMa, which was first identified in this laboratory in rabbit nasal microsomes, is now known to represent two distinct gene p roducts, P450s 2A10 and 2A11. In the present study; chimeric and site- directed mutants of 2A11 were constructed to determine which of the ei ght different amino acid residues are responsible for the much greater activity of 2A10 toward coumarin and testosterone. Mutation of Arg(62 ) and Asp(63) of 2A11 to the corresponding residues in 2A10, or mutati on of Thr(120) to Ser, as found in 2A10, did not change the activities . However, mutation of Arg(62), Asp(63), Gln(104), Ala(117), and Thr(1 20) of 2A11 to the corresponding residues in 2A10 resulted in a protei n that is as active as 2A10 in coumarin hydroxylation and approximatel y half as active as 2A10 in androstenedione formation. Mutation of Arg (372) in 2A11 to His, as found in 2A10, resulted in a significant incr ease in the rate of hydroxylation of testosterone, but not of coumarin . Our findings indicate that the identity of the amino acid at positio n 104 and/or 117 is important for activity with testosterone and for r egioselectivity at the 17 position, as well as for optimal activity wi th coumarin. In contrast, the identity of the residue at position 372 is important for optimal activity with testosterone but not the regios electivity at the 17 position and does not influence the activity with coumarin. (C) 1994 Academic Press, Inc.