Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol.
To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous 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 indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-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 Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, 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 increased 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 mutations we suggest that cholesterol enters P450 7A1 through the membrane, and 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 7A1-membrane interactions and the mechanism for substrate specificity.
Nakayama
K
K
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031, USA.
Puchkaev
A
A
Pikuleva
I A
IA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D002784:Cholesterol / Q000378:metabolism*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D002784:Cholesterol; D002790:Cholesterol 7-alpha-Hydroxylase