To gain further insight into the structure/function relationship of cytochrome P450 side-chain cleavage (CYP11A1), this enzyme was investigated in the Cape baboon (Papio ursinus). Four constructs were cloned and characterised in non-steroidogenic mammalian COS-1 cells. Wild type recombinant baboon CYP11A1 cDNA yielded a K(m) value of 1.6 microM for 25-hydroxycholesterol. The single amino acid substitutions, I98Q and I98K resulted in a 1.7- and 2.8-fold increases in K(m) values, respectively. Conversely, the introduction of the mutation, K103A, resulted in a 1.8-fold decrease in K(m). A homology model of CYP11A1, based on the crystal structures of CYP102 and CYP2C5, revealed that residues 98 and 103 lie within the B'-C loop and contribute to the spatial orientation and structural integrity of this domain. Based on these results we propose a topological model of the CYP11A1 active pocket, which is supported by substrate docking analysis and kinetic studies.