Purification and properties of a shortened form of cytochrome P-450 2E1: deletion of the NH2-terminal membrane-insertion signal peptide does not alter the catalytic activities.
As reported previously, alcohol-inducible cytochrome P-450 2E1 lacking the hydrophobic NH2-terminal segment is located primarily in the inner cell membrane when expressed in Escherichia coli and is active with a typical substrate. To study the catalytic properties in detail, we have purified the truncated P-450 lacking residues 3-29 to electrophoretic homogeneity from the solubilized bacterial membrane fraction in the presence of 4-methylpyrazole as a stabilizing agent. The resulting heme protein with a specific content of 15.8 nmol of P-450 per mg of protein has a reduced CO difference spectrum identical to that of the full-length enzyme, with a Soret maximum at 452 nm. The rates of catalysis of four reactions in the reconstituted enzyme system, including the oxygenation of ethanol to give acetaldehyde, the oxidative dealkylation of N-nitrosodiethylamine to give ethylene and acetaldehyde, and the ring hydroxylation of aniline and p-nitrophenol, are the same with the shortened and full-length enzymes. The apparent Km of p-nitrophenol is also the same, as is that for NADPH-cytochrome P-450 reductase and for cytochrome b5, which stimulates p-nitrocatechol formation about 3-fold. Moreover, the requirement for phosphatidylcholine for full catalytic activity is unchanged despite the absence of the NH2-terminal segment. Although this highly hydrophobic segment is believed to play a role in the intact cell as a membrane-insertion signal sequence, we conclude that it has no function in the catalytic activity of the cytochrome as an oxygenase, including interactions with the other components of the enzyme system.
Larson
J R
JR
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
Coon
M J
MJ
Porter
T D
TD
D000595:Amino Acid Sequence; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D002872:Chromosome Deletion; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D006882:Hydroxyapatites; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D009251:NADPH-Ferrihemoprotein Reductase