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Prot Cell    2011, Vol. 2 Issue (8) : 656-671    https://doi.org/10.1007/s13238-011-1082-6      PMID: 21904981
RESEARCH ARTICLE
Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)
Benjamin Rowlatt, Jake A. Yorke, Anthony J. Strong, Christopher J. C. Whitehouse, Stephen G. Bell, Luet-Lok Wong()
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK
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Abstract

Fatty acid binding and oxidation kinetics for wild type P450BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than kcat. The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.
Keywords P450BM3      monooxygenase      fatty acid      cooperativity      allosteric effect      CYP102A1     
Corresponding Author(s): Wong Luet-Lok,Email:luet.wong@chem.ox.ac.uk   
Issue Date: 01 August 2011
 Cite this article:   
Benjamin Rowlatt,Jake A. Yorke,Anthony J. Strong, et al. Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)[J]. Prot Cell, 2011, 2(8): 656-671.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1082-6
https://academic.hep.com.cn/pac/EN/Y2011/V2/I8/656
50 mmol/L TrisWith added Tris (mmol/L)With added phosphate (mmol/L)With added KCl (mmol/L)
100200300400100200300400100200300400
Laurate40455060654555606545506065
Myristate65707580806570758065707575
Palmitate95959595959595959595959595
Tab.1  Percentage of high spin heme content (estimated to within 5%) of fatty acid-bound full-length P450 at 30°C, pH 7.4, in different buffers and salt concentrations
Fig.1  Time courses at 30°C, pH 7.4 for Fe-CO complex formation monitored at 450 nm for P450.
(A) Laurate in 50 mmol/L Tris, which required a single exponential ( = 53.1s) to fit the data. (B) Laurate in 100 mmol/L phosphate, which required a double exponential to fit contributions from the fast phase ( = 108s) and a denatured phase ( = 30s), the fast phase accounting for 88% of the amplitude. (C) Myristate in 50 mmol/L Tris (single exponential fit: = 98s). (D) Myristate in 100 mmol/L phosphate (double exponential fit: = 226s, 56%; = 33s, 44%). (E) Palmitate in 50 mmol/L Tris (double exponential fit: = 218s, 52%; = 31s, 48%). (F) Palmitate in 100 mmol/L phosphate (double exponential fit: = 306s, 41%; = 37s, 59%).
Fig.1  Time courses at 30°C, pH 7.4 for Fe-CO complex formation monitored at 450 nm for P450.
(A) Laurate in 50 mmol/L Tris, which required a single exponential ( = 53.1s) to fit the data. (B) Laurate in 100 mmol/L phosphate, which required a double exponential to fit contributions from the fast phase ( = 108s) and a denatured phase ( = 30s), the fast phase accounting for 88% of the amplitude. (C) Myristate in 50 mmol/L Tris (single exponential fit: = 98s). (D) Myristate in 100 mmol/L phosphate (double exponential fit: = 226s, 56%; = 33s, 44%). (E) Palmitate in 50 mmol/L Tris (double exponential fit: = 218s, 52%; = 31s, 48%). (F) Palmitate in 100 mmol/L phosphate (double exponential fit: = 306s, 41%; = 37s, 59%).
kf (s-1) in phosphate (mmol/L)kf (s-1) in Tris (mmol/L)
102510020030050100250
Laurate––107±21117±8118±1258±7.651±9.850±3.7
Myristate––223±23––102±7––
Palmitate234±7305±19303±24288±23279±24227±15218±33217±39
Tab.2  Values of (s), the rate constant for the formation of the Fe-CO form of P450 complexed with laurate, myristate and palmitate in different concentrations of Tris and phosphate buffers (30°C, pH 7.4)
Fig.2  Kinetic titrations of P450 at 30°C, pH 7.4.
(A) With laurate in 50 mmol/L Tris and 100 mmol/L phosphate. (B) With myristate in 50 mmol/L Tris. (C) With palmitate in 50 mmol/L Tris. (D) With palmitate in 100 mmol/L phosphate. The enzyme concentration was 0.1 μmol/L. All data were fitted to hyperbolic functions except for (C) where a Hill equation with an intercept was used.
Fig.2  Kinetic titrations of P450 at 30°C, pH 7.4.
(A) With laurate in 50 mmol/L Tris and 100 mmol/L phosphate. (B) With myristate in 50 mmol/L Tris. (C) With palmitate in 50 mmol/L Tris. (D) With palmitate in 100 mmol/L phosphate. The enzyme concentration was 0.1 μmol/L. All data were fitted to hyperbolic functions except for (C) where a Hill equation with an intercept was used.
100 mmol/L phosphate50 mmol/L Tris
kcat (s-1)KM/H (μmol/L)nkcat (s-1)KM/H (μmol/L)n
Laurate82±2.0170±12173±2.5239±211
Myristate138±3.170±2.7194±3.479±5.01.2
Palmitate122±5.983±9.0187±1.452±1.04.8±0.3*
Tab.3  Apparent kinetic parameters for the oxidation of laurate, myristate and palmitate in 50 mmol/L Tris and 100 mmol/L phosphate (30°C, pH 7.4) with the enzyme concentration at 0.1 μmol/L
Fig.3  Kinetic titrations of P450.
P450 (0.1 μmol/L) with palmitate at pH 7.4, 30°C, showing the onset of sigmoidal behaviour at 50 mmol/L phosphate (A–C), and in 10 mmol/L phosphate (E) with added ammonium sulfate (D) and sodium carbonate (F) showing the effect of ionic strength. The data for low phosphate buffer concentrations, in panels (C–F), were fitted to a modified Hill equation with an intercept; black lines depict best fits where the intercept was allowed to vary, while fits using the background NADPH consumption rate of 0.5 s in the absence of substrate as the intercept are shown as red dashed lines.
Fig.3  Kinetic titrations of P450.
P450 (0.1 μmol/L) with palmitate at pH 7.4, 30°C, showing the onset of sigmoidal behaviour at 50 mmol/L phosphate (A–C), and in 10 mmol/L phosphate (E) with added ammonium sulfate (D) and sodium carbonate (F) showing the effect of ionic strength. The data for low phosphate buffer concentrations, in panels (C–F), were fitted to a modified Hill equation with an intercept; black lines depict best fits where the intercept was allowed to vary, while fits using the background NADPH consumption rate of 0.5 s in the absence of substrate as the intercept are shown as red dashed lines.
kcat (s-1)KM/H (μmol/L)nvo (s-1)
10 mmol/L32±1.031±1.34.011
25 mmol/L56±1.150±1.34.811
50 mmol/L98±6.665±7.21.4–
75 mmol/L116±6.989±111–
100 mmol/L122±5.983±9.01–
10 mmol/L+ 15 mmol/L Na2CO3 + 15 mmol/L (NH4)2SO440±0.735±1.34.35
72±1.868±2.63.720
25 mmol/L+ 50 mmol/L Tris95±3.362±3.02.316
Tab.4  Apparent kinetic parameters for the oxidation of palmitate by P450 in different phosphate buffer concentrations and in mixtures with other compounds
Fig.4  Plots of peak-to-trough separations in the difference spectra for binding titrations for the heme domain of P450 at 30°C, pH 7.4.
(A) Laurate in 50 mmol/L Tris and 100 mmol/L phosphate; the enzyme concentration in both buffers was 0.5 μmol/L. (B) Myristate in 50 mmol/L Tris at an enzyme concentration of 5 μmol/L. The laurate binding data were fitted to a rectangular hyperbola. The data for myristate binding showed a slight kink in the 10-12 μmol/L concentration range but gave an acceptable fit to a hyperbolic function.
Fig.4  Plots of peak-to-trough separations in the difference spectra for binding titrations for the heme domain of P450 at 30°C, pH 7.4.
(A) Laurate in 50 mmol/L Tris and 100 mmol/L phosphate; the enzyme concentration in both buffers was 0.5 μmol/L. (B) Myristate in 50 mmol/L Tris at an enzyme concentration of 5 μmol/L. The laurate binding data were fitted to a rectangular hyperbola. The data for myristate binding showed a slight kink in the 10-12 μmol/L concentration range but gave an acceptable fit to a hyperbolic function.
Fig.5  Palmitate binding titrations with P450 at 30°C, pH 7.4 showing the plot of peak-to-trough separations ( - ), scaled for dilution.
(A) For the heme domain (1 μmol/L) in 100 mmol/L phosphate. (B) For the heme domain (5 μmol/L) in 50 mmol/L Tris. (C) For the full-length enzyme (2.5 μmol/L) in 50 mmol/L Tris. (D) For the heme domain (5 μmol/L) in 10 mmol/L phosphate. The data in (A) were fitted to a two-site binding model while the complex behaviour in (B–D) required a four-site binding model.
Fig.5  Palmitate binding titrations with P450 at 30°C, pH 7.4 showing the plot of peak-to-trough separations ( - ), scaled for dilution.
(A) For the heme domain (1 μmol/L) in 100 mmol/L phosphate. (B) For the heme domain (5 μmol/L) in 50 mmol/L Tris. (C) For the full-length enzyme (2.5 μmol/L) in 50 mmol/L Tris. (D) For the heme domain (5 μmol/L) in 10 mmol/L phosphate. The data in (A) were fitted to a two-site binding model while the complex behaviour in (B–D) required a four-site binding model.
Fig.6  Difference spectra for the binding titration of palmitic acid with the P450 heme domain in 50 mmol/L Tris, pH 7.4 at 30°C.
The protein concentration was 5 μmol/L. The difference spectra in the presence of various total concentrations of palmitic acid, added from stock solutions in DMSO, are shown. The total concentration of DMSO added was 2% (). Inset shows the plot of the peak-to-trough difference ( - ), scaled for dilution, against the total substrate concentration.
Fig.6  Difference spectra for the binding titration of palmitic acid with the P450 heme domain in 50 mmol/L Tris, pH 7.4 at 30°C.
The protein concentration was 5 μmol/L. The difference spectra in the presence of various total concentrations of palmitic acid, added from stock solutions in DMSO, are shown. The total concentration of DMSO added was 2% (). Inset shows the plot of the peak-to-trough difference ( - ), scaled for dilution, against the total substrate concentration.
Fig.7  Difference spectra for binding titrations at 30°C, pH 7.4 of palmitic acid with P450.
(A) The heme domain (5 μmol/L) in 10 mmol/L phosphate, and (B) the full-length enzyme (2.5 μmol/L) in 50 mmol/L Tris. Palmitic acid was added from stock solutions in DMSO and the total concentration of DMSO added was 2% ().
Fig.7  Difference spectra for binding titrations at 30°C, pH 7.4 of palmitic acid with P450.
(A) The heme domain (5 μmol/L) in 10 mmol/L phosphate, and (B) the full-length enzyme (2.5 μmol/L) in 50 mmol/L Tris. Palmitic acid was added from stock solutions in DMSO and the total concentration of DMSO added was 2% ().
K1 (μmol/L) ΔA1K2 (μmol/L) ΔA2K3 (μmol/L) ΔA3K4 (μmol/L) ΔA4
Heme domain P450BM3
10 mmol/L phosphate6.15±1.04.70±0.07320±251.61±0.22
0.63±0.070.09±0.020.10±0.010.41±0.02
50 mmol/L Tris6.50±0.5010.0±1.51380±301.80±0.25
0.92±0.080.10±0.010.05±0.010.56±0.01
Full-length P450BM3
50 mmol/L Tris1.20±0.0810.5±0.15128±102.0±0.17
0.27±0.020.14±0.020.10±0.010.25±0.01
Tab.5  Palmitate binding data for P450 in Tris and phosphate buffers
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