Autocatalytic mechanism and consequences of covalent heme attachment in the cytochrome P4504A family.
The prosthetic heme group in the CYP4A family of cytochrome P450 enzymes is covalently attached to an I-helix glutamic acid residue. This glutamic acid is conserved in the CYP4 family but is absent in other P450 families. As shown here, the glutamic acid is linked, presumably via an ester bond, to a hydroxyl group on the heme 5-methyl group. Mutation of the glutamic acid to an alanine in CYP4A1, CYP4A3, and CYP4A11 suppresses covalent heme binding. In wild-type CYP4A3 68% of the heme is covalently bound to the heterologously expressed protein, but in the CYP4A3/E318D mutant, 47% of the heme is unchanged, 47% is present as noncovalently bound 5-hydroxymethylheme, and only 6% is covalently bound to the protein. In the CYP4A3/E318Q mutant, the majority of the heme is unaltered, and <2% is covalently linked. The proportion of covalently bound heme in the recombinant CYP4A proteins increases with time under turnover conditions. The catalytic activity is sensitive in some, but not all, CYP4A enzymes to the extent of covalent heme binding. Mutations of Glu(318) in CYP4A3 decrease the apparent k(cat) values for lauric acid hydroxylation. The key conclusions are that (a) covalent heme binding occurs via an ester bond to the heme 5-methyl group, (b) covalent binding of the heme is mediated by an autocatalytic process, and (c) fatty acid oxidation is sensitive in some CYP4A enzymes to the presence or absence of the heme covalent link.
LeBrun
Laurie A
LA
Department of Pharmaceutical Chemistry, School of Pharmacy S-926, University of California-San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0446, USA.
Hoch
Ute
U
Ortiz de Montellano
Paul R
PR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006418:Heme / Q000737:chemistry*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D006418:Heme; D003577:Cytochrome P-450 Enzyme System