Characterization and functional analysis of two common human cytochrome P4501B1 variants

Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates pr ocarinogens, metabolizes 17 beta-estradiol, and may well have a role in the pathogenesis of some forms of cancer, Besides rare deleterious mutations r eported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges, We have expressed two o f the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in ord er to functionally characterize the alleles with respect to kinetic propert ies and protein stability. The CYP1B1.2 variant contains the two linked ami no acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic para meters of two structurally unrelated CYP1B1 substrates for the two variants were examined, No kinetic differences were seen of 17 beta-estradiol hydro xylation activities between the two CYP1B1 variants and an only minor incre ase in the apparent K-m for ethoxyresorufin was observed for CYP1B1.2. It t herefore appears that they have very similar catalytic properties and the s ubstitutions do not appear to alter CYP1B1 catalytic function. The two CYP1 B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stab ility. The combined results indicate that these two CYP1B1 variants show ve ry similar properties with respect to catalytic activities and protein stab ility and do not alter CYP1B1 function. (C) 2000 Academic Press.