Hybrid cytochromes P-450 identify a substrate binding domain in P-450IIC5 and P-450IIC4.
The cytochrome P-450 superfamily of enzymes catalyzes the oxidative metabolism of innumerable lipophilic compounds (e.g., drugs, carcinogens, steroids). Although the three-dimensional structure of a soluble bacterial P-450 (P-450cam) has been solved, little is known about the structures of the membrane-bound mammalian P-450s. Thus, the structural features of these enzymes that determine their multisubstrate specificity are unknown. In this report, we identify a segment of the primary structure of the structurally similar but functionally distinct cytochromes P-450IIC5 and P-450IIC4, which determines the apparent affinity of these cytochromes for the conversion of progesterone into the mineralocorticoid deoxycorticosterone. P-450IIC5 exhibits a greater than 10-fold lower apparent Km than P-450IIC4 for progesterone 21-hydroxylation. Chimeric cDNAs were constructed and expressed in COS-1 cells, which encode hybrids between these enzymes. The hybrid enzymes were assayed for catalytic activity and compared to the parental proteins. A segment of P-450IIC5 was identified that conferred the lower Km of P-450IIC5 to P-450IIC4. Sequential reduction of the length of the exchanged segments led to a hybrid enzyme with a high affinity derived largely from P-450IIC4, which contains three amino acid residues derived from P-450IIC5 clustered between positions 113 and 118. This suggests that this region is part of a substrate binding domain. This region maps by alignment of amino acid sequences to a residue of P-450cam, which has been implicated in substrate binding, suggesting that these segments of the primary structure serve a similar functional role in these two distantly related proteins.
Kronbach
T
T
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Larabee
T M
TM
Johnson
E F
EF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002678:Chimera; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D005796:Genes; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D014162:Transfection
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013255:Steroid 21-Hydroxylase