Membrane binding and substrate access merge in cytochrome P4507Al, a key enzyme in degradation of cholesterol

To study membrane topology and mechanism for substrate specificity, we trun cated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introd uced conservative and nonconservative substitutions at positions 214-227. H eterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indic ate that a hydrophobic region, comprising residues 214-227, forms a seconda ry site of attachment to the membrane in P450 7A1 in addition to the NH2-te rminal signal-anchor sequence. There are two groups of residues at this enz yme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224 , and Phe227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L 218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K-m for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was i ncreased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mu tations we suggest that cholesterol enters P450 7A1 through the membrane, a nd Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7 A1membrane interactions and the mechanism for substrate specificity.