Phosphorylation of cytochrome P450: Regulation by cytochrome b5

• Published in: Archives of biochemistry and biophysics Vol. 271; no. 2; pp. 424 - 432
• Main Authors: Epstein, Paul M., Curti, Mario, Jansson, Ingela, Huang, Chi-Kuang, Schenkman, John B.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.06.1989; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Binding, Competitive; Biological and medical sciences; Cyclic AMP - pharmacology; Cytochrome b Group - pharmacology; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - metabolism; Cytochromes b5; Fundamental and applied biological sciences. Psychology; Hemoproteins; Hydrogen-Ion Concentration; Kinetics; Metalloproteins; Microsomes, Liver - enzymology; Phosphorylation; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Protein Kinases - metabolism; Proteins; Rats; Hemoprotein; Rat; Enzyme; Liver; Rodentia; Rabbit; Lagomorpha; Vertebrata; Regulation(control); Mammalia; Enzymatic activity; Kinetics; Ultraviolet visible spectrometry
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(89)90292-0

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441–448) that cytochrome b 5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b 5 is competitive, with a K i = 0.48 μM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b 5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b 5 binding domain of the enzymes.