Unique Properties of Purified, Escherichia coli-Expressed Constitutive Cytochrome P4504A5

Author： Hosny G. Roman L.J. Mostafa M.H. Masters B.S.S.

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.366, Iss.2, 1999-06, pp. : 199-206

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Abstract

Cytochromes P450 of the 4A family metabolize a variety of fatty acids, prostaglandins, and eicosanoids mainly at the terminal carbon (ω-hydroxylation) and, to a lesser extent, at the penultimate carbon [(ω-1)-hydroxylation]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of ∼80 nmol/liter of cells. Spectroscopic characterization of the purified enzyme, using electron paramagnetic resonance and absolute and substrate-perturbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrate binding. The k_cat and K_m values for laurate ω-hydroxylation were 41 min^-1 and 8.5 μM, respectively, in the absence of cytochrome b5, and 138 min^-1 and 38 μM, respectively, in the presence of cytochrome b5. Hydroxylation of palmitate was dependent on the presence of cytochrome b5; k_cat and K_m values were 48 min^-1 and 122 μM, respectively. Hydroxylation of arachidonic acid was barely detectable and was unchanged by the addition of cytochrome b5.