SITE-DIRECTED MUTAGENESIS OF MOUSE STEROID 7-ALPHA-HYDROXYLASE (CYTOCHROME-P-4507-ALPHA) - ROLE OF RESIDUE-209 IN DETERMINING STEROID CYTOCHROME-P-450 INTERACTION

We have cloned a cDNA encoding mouse steroid 7alpha-hydroxylase P450(7 alpha) (cytochrome P-450(7alpha) and expressed it in Saccharomyces cer evisiae. Mouse P450(7alpha) is 70 % identical in its amino acid sequen ce with the mouse steroid 15alpha-hydroxylase P450(15alpha) (2A4). The Leu at position 209 of P450(15alpha) is the most important residue to determine the steroid hydroxylase activity of the P450 [Lindberg and Negishi (1989) Nature (London) 339, 632-634]. The P450(7alpha) contain s Asn at the position corresponding to the Leu-209 of P450(15alpha), a lthough both P450s hydroxylate testosterone. The CO-reduced P450(7alph a) complex is unstable, so that it is quickly converted into the inact ive P420, whereas the P450(15alpha) is very stable. The P450(7alpha), however, is stabilized either by addition of testosterone or by a muta tion of Asn-209 to Leu. The mutant P450(7alpha) displays a 17-fold low er V(max) value than the wild-type enzyme. Unexpectedly, it also has 3 -fold lower K(m) and K(d) values. Residue 209 in P450(7alpha), therefo re, appears to be located at a critical site of the haem-substrate-bin ding pocket. Corticosterone inhibits the testosterone 7alpha-hydroxyla se activity of the wild-type P450(7alpha), whereas it does not inhibit the mutant P450(7alpha). Conversely, the P450(15alpha) activity becom es inhibited by corticosterone upon the replacement of Leu-209 by Asn. In addition, this mutation increases the corticosterone 15alpha-hydro xylase activity of P450(15alpha) at least 20-fold. Whereas the inhibit ion by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209. The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificit y of steroid binding to both P450(7alpha) and P450(15alpha).