The kinetic and spectral characterization of the E. coli-Expressed mammalian CYP4A7: Cytochrome b5 effects vary with substrate

Patricia A. Loughran, Linda J. Roman, R. Timothy Miller, Bettie Sue S. Masters

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins. CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E. coli by adding the first 10 codons of CYP17α producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies. CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min-1 and corresponding Km values of 74.5, 27, and 16.7 μM, respectively, in the presence of cytochrome b5. In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min-1 and corresponding Km values of 3.9 and 33 μM, respectively. Arachidonate was not metabolized in the absence of cytochrome b5. Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min-1 and Km values of 74 and 25 μM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role. Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (Ks) in the absence and presence of cytochrome b5 (13 and 43 μM, respectively). In stopped-flow kinetics experiments, the flavin reduction (∼90 s-1) and heine reduction (∼9 s-1) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5. Estimations of the rate of CPR (0.3 s-1) or cytochrome b5 (9.1 s-1) binding with CYP4A7 were also determined.

Original languageEnglish (US)
Pages (from-to)311-321
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume385
Issue number2
DOIs
StatePublished - Jan 15 2001

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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