Cassette mutagenesis was used to analyze the effects of mutations at amino acid positions 107-120 in cytochrome P450 2C2 which are part of a predicted substrate recognition site, SRS-1 (Gotoh, O. (1992) J. Biol. Chem. 267, 83- 90), and terminate with a highly conserved tryptophan. All the mutant enzymes were expressed in COS1 cells at approximately the same level as wild type as determined by immunoprecipitation of radiolabeled products. Substitutions of amino acids from 107 to 110 and from 116 to 119 resulted in mutant enzymes that retained substantial lauric acid hydroxylase activity suggesting that these residues are not critical determinants of substrate specificity. In contrast, amino acids 112-115 were characterized by strong decreases in activity in at least one mutation tested. A substitution of phenylalanine for valine 112 reduced activity about 3-fold. At position 113, substitution of leucine for isoleucine had little effect, but activity was reduced 15-fold by substitution with cysteine. A conservative mutation at position 114 reduced activity 5-fold, a nonconservative mutation at position 115 resulted in a 30- fold reduction of lauric acid hydroxylation. Substitution of glutamic acid for glycine at position 109 had little effect on activity, while more conservative substitutions of valine for glycines at positions 111 and 117 resulted in about 50- and 7-fold reductions of activity, respectively. Substitution of leucine for tryptophan 120 resulted in a 7-fold reduction of activity indicating that tryptophan at this position contributes to, but is not essential for, enzymatic activity in spite of its presence in nearly all eukaryotic cytochromes P450. The results are consistent with a model in which amino acids 112-114, and possibly 115, align with a loop of bacterial P450cam sequence containing substrate contacting amino acids, tyrosine 96 and phenylalanine 98. Glycines at either end of the loop in cytochrome P450 2C2 appear to be critical for activity.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology