Structure-function studies of human aromatase by site-directed mutagenesis: kinetic properties of mutants Pro-308----Phe, Tyr-361----Phe, Tyr-361----Leu, and Phe-406----Arg.
Aromatase, a cytochrome P450, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [1 beta,2 beta-3H]androstenedione, [19-14C]androstenedione, and [1 beta,2 beta-3H]testosterone as substrates. The mutant Phe-406----Arg was completely inactive. Since there were only small changes in the Km and Vmax values for all substrates for mutants Tyr-361----Phe and Tyr-361----Leu, the residue Tyr-361 appears not to be directly involved in the substrate binding. The mutant Pro-308----Phe had altered catalytic properties; the Km values for androstenedione, but not testosterone, decreased significantly. These results, along with those obtained from inhibition studies with aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide, suggest that Pro-308 is probably situated in the active site of the enzyme and may be interacting with the D ring of the steroids.
Zhou
D J
DJ
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Pompon
D
D
Chen
S A
SA
D000595:Amino Acid Sequence; D000616:Aminoglutethimide / Q000494:pharmacology; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001120:Arginine; D001141:Aromatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010649:Phenylalanine; D011392:Proline; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine
D015345:Oligonucleotide Probes; D000616:Aminoglutethimide; D013739:Testosterone; D000735:Androstenedione; D014443:Tyrosine; D010649:Phenylalanine; D001120:Arginine; D011392:Proline; D001141:Aromatase; D007930:Leucine