TY - JOUR
T1 - Unique properties of purified, Escherichia coli-expressed constitutive cytochrome P4504A5
AU - Hosny, Gihan
AU - Roman, Linda J.
AU - Mostafa, Mostafa H.
AU - Masters, Bettie Sue S.
N1 - Funding Information:
We thank Dr. John C. Salerno for his help in recording and interpreting the EPR spectra. We are grateful to Mr. Thomas M. Shea, in this laboratory, for supplying us with NADPH-cytochrome P450 reductase; to Dr. Timothy McCabe who, while in this laboratory, provided advice in running the HPLC assays; and to Dr. Pavel Martasek for his intellectual support in our studies. This work was supported by NIH Grant GM31296 to B.S.S.M. and an Egyptian Scholar Grant to B.S.S.M. for G.H.
PY - 1999/6/15
Y1 - 1999/6/15
N2 - 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.
AB - 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.
KW - 4A5
KW - Cytochrome P450
KW - Fatty acid hydroxylase
KW - Lauric acid
KW - P450
KW - P4504A5
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U2 - 10.1006/abbi.1999.1214
DO - 10.1006/abbi.1999.1214
M3 - Article
C2 - 10356284
AN - SCOPUS:0033563820
VL - 366
SP - 199
EP - 206
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 2
ER -