Calibration of the channel that determines the omega-hydroxylation regiospecificity of cytochrome P4504A1: catalytic oxidation of 12-HALODOdecanoic acids.
The fatty acid omega-hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in H(2)(18)(2)O show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O(2). The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an omega-1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the omega-aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[(2)H](2)-12-chlorododecanoic acid causes a 2-3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl >> I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 Angstroms but smaller than 4.30 Angstroms.
He
Xiang
X
Department of Pharmaceutical Chemistry, University of California, San Francisco, 94143, USA.
Cryle
Max J
MJ
De Voss
James J Ortiz
JJ
de Montellano
Paul R
PR
D000447:Aldehydes / Q000737:chemistry; D000818:Animals; D001966:Bromine / Q000737:chemistry*; D002138:Calibration; D002244:Carbon / Q000737:chemistry; D002713:Chlorine / Q000737:chemistry*; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D000072470:Cytochrome P450 Family 4; D005227:Fatty Acids / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D006900:Hydroxylation; D007455:Iodine / Q000737:chemistry*; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry*; D013058:Mass Spectrometry; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013053:Spectrophotometry; D013237:Stereoisomerism; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays; D014867:Water / Q000737:chemistry
D000447:Aldehydes; C465387:Cyp4a1protein, rat; D005227:Fatty Acids; D007850:Lauric Acids; D014867:Water; D002713:Chlorine; D002244:Carbon; D003577:Cytochrome P-450 Enzyme System; D007455:Iodine; D019392:Cytochrome P-450 CYP2E1; D000072470:Cytochrome P450 Family 4; C034879:12-bromododecanoic acid; D010100:Oxygen; D001966:Bromine