Difference between revisions of "Transformation of AA to 15-HPETE"

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[[Welcome to the In-Silico Model of Cutaneous Lipids Wiki | Return to overview]]
 
[[Welcome to the In-Silico Model of Cutaneous Lipids Wiki | Return to overview]]
 +
 +
The genes ALOX15B and ALOX15 encodes the formation of two isoforms of 15-lioxygenase (15-LOX). The gene ALOX15B encodes the formation of 15-LOX-2, which converts AA exclusively into 15(S)-HPETE.  Whereas the gene ALOX15 encodes the 15-LOX-1 protein, which converts AA into both 12(S)-HPETE and 15(S)-HPETE. The formation of the unstable hydroperoxy fatty acids (HPETE) begins with the abstraction of a hydrogen radical at the allylic position between two double bonds. The structure undergoes a rearrangement reaction which results in the formation of a conjugated diene system. The insertion of molecular oxygen and a hydrogen leads to the formation of the final structure, a hydroperoxy fatty acid.
 
== Reaction ==
 
== Reaction ==
  
Line 6: Line 8:
 
==Chemical equation==
 
==Chemical equation==
  
−
<center><math> AA \rightleftharpoons PGH2 </math></center>
+
<center><math> AA \rightleftharpoons 15-HPETE </math></center>
  
 
== Rate equation ==
 
== Rate equation ==
  
 +
[[File:R17.PNG|center|500px]]
  
 
== 15-LOX Parameters ==
 
== 15-LOX Parameters ==
−
 
+
===K<sub>ms</sub>===
 
{|class="wikitable sortable"  
 
{|class="wikitable sortable"  
−
|+  style="text-align: left;" | Michaelis-Menten Constants
+
|+  style="text-align: left;" | Literature values
 
|-
 
|-
 
! Value
 
! Value
Line 20: Line 23:
 
! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
Line 25: Line 29:
 
|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Epithelium
+
|Expression Vector: Epithelium
 +
Enzyme: 15-Lipoxygenase
 +
pH: 7.5
 +
Temperature: 25
 +
|512
 
|<ref name="Jacquot2008"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Jacquot C. "Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.'' Biochemistry. 2008 Jul 8;47(27):7295-303. doi: 10.1021/bi800308q. Epub 2008 Jun 12.]</ref>  
 
|<ref name="Jacquot2008"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Jacquot C. "Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.'' Biochemistry. 2008 Jul 8;47(27):7295-303. doi: 10.1021/bi800308q. Epub 2008 Jun 12.]</ref>  
 
|-
 
|-
Line 31: Line 39:
 
|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Reticulocyte
+
|Expression Vector: Reticulocyte
 +
Enzyme: 15-Lipoxygenase
 +
pH:7.5
 +
Temperature: Unspecified
 +
|512
 
|<ref name="Jacquot2008b"> [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883171/ Jacquot C. "Synthesis of 11-Thialinoleic Acid and 14-Thialinoleic Acid, Inhibitors of Soybean and Human Lipoxygenases'' Org Biomol Chem. 2008 Nov 21; 6(22): 4242–4252.]</ref>  
 
|<ref name="Jacquot2008b"> [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883171/ Jacquot C. "Synthesis of 11-Thialinoleic Acid and 14-Thialinoleic Acid, Inhibitors of Soybean and Human Lipoxygenases'' Org Biomol Chem. 2008 Nov 21; 6(22): 4242–4252.]</ref>  
 
|-
 
|-
Line 37: Line 49:
 
|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Keratinocyte
+
|Expression Vector: Keratinocyte
 +
Enzyme: 15-Lipoxygenase
 +
pH: 6.7 - 7.3
 +
Temperature: 37
 +
|2048
 
|<ref name="Burrall1988"> [http://www.ncbi.nlm.nih.gov/pubmed/2459258 Burrall B. "Enzymatic properties of the 15-lipoxygenase of human cultured keratinocytes.'' J Invest Dermatol. 1988 Oct;91(4):294-7.]</ref>  
 
|<ref name="Burrall1988"> [http://www.ncbi.nlm.nih.gov/pubmed/2459258 Burrall B. "Enzymatic properties of the 15-lipoxygenase of human cultured keratinocytes.'' J Invest Dermatol. 1988 Oct;91(4):294-7.]</ref>  
 
|-
 
|-
Line 43: Line 59:
 
|<math> mM </math>
 
|<math> mM </math>
 
|Human
 
|Human
−
| Wildtype 15-Lipoxygenase
+
|Expression Vector: Baculovirus Insect Cell
 +
Enzyme: Wildtype 15-Lipoxygenase
 +
pH: 6.8
 +
Temperature: 37
 +
|512
 
|<ref name="Sloane1995"> [http://www.ncbi.nlm.nih.gov/pubmed/7479689 Sloane D. L. "Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.'' Protein Eng. 1995 Mar;8(3):275-82..]</ref>  
 
|<ref name="Sloane1995"> [http://www.ncbi.nlm.nih.gov/pubmed/7479689 Sloane D. L. "Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.'' Protein Eng. 1995 Mar;8(3):275-82..]</ref>  
 
|-
 
|-
 
|}
 
|}
  
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 15-LOX Kms distribution
 +
! Mode (mM) !! Confidence Interval !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 1.02E-02 || 2.40E+00 || -4.42E+00 || 4.10E-01
 +
|-
 +
|}
 +
 +
[[Image:57.jpg|none|thumb|500px|The estimated probability distribution for 15-LOX Kms. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
 +
 +
 +
===K<sub>mp</sub>===
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 15-LOX Kmp distribution
 +
! Mode (mM) !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 1.02E-02 || -4.42E+00 || 4.11E-01
 +
|}
 +
 +
[[Image:58.jpg|none|thumb|500px|The estimated probability distribution for 15-LOX Kmp. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
 +
 +
===k<sub>cat</sub>===
 
{|class="wikitable sortable"  
 
{|class="wikitable sortable"  
−
|+  style="text-align: left;" | Enzyme Turnover Numbers
+
|+  style="text-align: left;" | Literature values
 
|-
 
|-
 
! Value
 
! Value
Line 55: Line 97:
 
! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
Line 60: Line 103:
 
|<math> minute^{-1} </math>
 
|<math> minute^{-1} </math>
 
|Human  
 
|Human  
−
|Epithelium
+
|Expression Vector: Epithelium
 +
Enzyme: 15-Lipoxygenase
 +
pH: 7.5
 +
Temperature: 25
 +
|512
 
|<ref name="Jacquot2008"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Jacquot C. "Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.'' Biochemistry. 2008 Jul 8;47(27):7295-303. doi: 10.1021/bi800308q. Epub 2008 Jun 12.]</ref>  
 
|<ref name="Jacquot2008"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Jacquot C. "Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.'' Biochemistry. 2008 Jul 8;47(27):7295-303. doi: 10.1021/bi800308q. Epub 2008 Jun 12.]</ref>  
 
|-
 
|-
 
|37.2 ± 1.8
 
|37.2 ± 1.8
 
|rowspan="7"|per minute
 
|rowspan="7"|per minute
−
|rowspan="7"|Human prostate epithelial 15-lipoxygenase-2
+
|rowspan="7"|Expression Vector:Human prostate epithelial  
−
|pH 7
+
Enzyme:15-lipoxygenase-2
−
|rowspan="7"|<ref name="Wecksler2009"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Jacquot C. "Kinetic and Structural Investigations of the Allosteric Site in Human Epithelial 15-Lipoxygenase-2'' Biochemistry, 2009, 48 (36), pp 8721–8730]</ref>  
+
|pH 7, Temperature: 22
 +
|rowspan="7"|512
 +
|rowspan="7"|<ref name="Wecksler2009"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Wecksler A. "Kinetic and Structural Investigations of the Allosteric Site in Human Epithelial 15-Lipoxygenase-2'' Biochemistry, 2009, 48 (36), pp 8721–8730]</ref>  
 
|-
 
|-
 
|45 ± 1.2
 
|45 ± 1.2
−
|pH 7.5
+
|pH 7.5, Temperature: 22
 
|-
 
|-
 
|44.4 ± 2.4
 
|44.4 ± 2.4
−
|pH 8
+
|pH 8, Temperature: 22
 
|-
 
|-
 
|34.2 ± 0.6
 
|34.2 ± 0.6
−
|15°C
+
|15°C, pH = 7.5
 
|-
 
|-
 
|45 ± 1.2
 
|45 ± 1.2
−
|22°C
+
|22°C, pH = 7.5
 
|-
 
|-
 
|62.4 ± 4.2
 
|62.4 ± 4.2
−
|30°C
+
|30°C, pH = 7.5
 
|-
 
|-
 
|82.8 ± 4.8
 
|82.8 ± 4.8
−
|37°C
+
|37°C, pH = 7.5
 +
|-
 +
|}
 +
 
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 15-LOX kcat distribution
 +
! Mode (min-1) !! Confidence Interval !! Location parameter (µ) !! Scale parameter (σ)
 
|-
 
|-
 +
| 6.51E+01 || 4.62E+00 || 4.60E+00 || 6.50E-01
 
|}
 
|}
 +
 +
[[Image:59.jpg|none|thumb|500px|The estimated probability distribution for 15-LOX kcat. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
 +
 +
===Enzyme concentration ===
 +
 +
To convert the enzyme concentration from ppm to mM, the following [[Common equations#Enzyme concentration (mM)|equation]] was used.
  
 
{|class="wikitable sortable"  
 
{|class="wikitable sortable"  
−
|+  style="text-align: left;" | Enzyme Concentrations
+
|+  style="text-align: left;" | Literature values
 
|-
 
|-
 
! Value
 
! Value
Line 96: Line 158:
 
! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
−
|1.60e-05 - 4.00e-05
+
|4.09
−
|mM
+
|<math> ppm </math>
−
|Human
+
|Human  
−
|Human reticulocyte 15-lipoxygenase-1, Enzymatic assays were performed in 25 mM Hepes buffer (pH 7.5,
+
|Expression Vector: Lung
−
25C), with and without product (12-HETE) addition, in the presence of 25 μM of substrate.
+
Enzyme: 15-LOX
−
|<ref name="Wecksler2009"> [www.ncbi.nlm.nih.gov/pubmed/19469483 Wecksler A. "Mechanistic Investigations of Human Reticulocyte 15- and Platelet 12-Lipoxygenases with Arachidonic Acid'' Biochemistry, 2009, 48 (26), pp 6259–6267]</ref>
+
pH: 7.5
 +
Temperature: 37 °C
 +
|1024
 +
|<ref name="Kim2014"> [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13302.pdf M. Kim ''A draft map of the human proteome'' Nature, 2014 509, 575–581]</ref>
 +
|-
 +
|37.4
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Spleen
 +
Enzyme: 15-LOX
 +
pH: 7.5
 +
Temperature: 37 °C
 +
|1024
 +
|<ref name="Wilhelm2014"> [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13319.pdf M. Wilhelm ''Mass-spectrometry-based draft of the human proteome'' Nature, 2014 509, 582–587]</ref>
 
|-
 
|-
−
|2.00e-05
+
|1.40
−
|mM
+
|<math> ppm </math>
 
|Human  
 
|Human  
−
|Epithelium
+
|Expression Vector: Gut
−
|<ref name="Jacquot2008"> [http://www.ncbi.nlm.nih.gov/pubmed/18547056 Jacquot C. "Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.'' Biochemistry. 2008 Jul 8;47(27):7295-303. doi: 10.1021/bi800308q. Epub 2008 Jun 12.]</ref> _
+
Enzyme: 12-LOX
 +
pH: 7.5
 +
Temperature: 37 °C
 +
|1024
 +
|<ref name="Kim2014"> [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13302.pdf M. Kim ''A draft map of the human proteome'' Nature, 2014 509, 575–581]</ref>
 +
|-
 +
|}
 +
 
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 15-LOX concentration distribution
 +
! Mode (ppm) !! Mode (mM) !! Confidence Interval !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 4.07 || 2.25E-05|| 7.23E+00 || 7.12E-01 || 1.19E+00
 +
|}
 +
 
 +
[[Image:157.jpg|none|thumb|500px|The estimated probability distribution for 15-LOX concentration. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.   ]]
 +
 
 +
===K<sub>eq</sub>===
 +
{|class="wikitable sortable"
 +
|+  style="text-align: left;" | Literature values
 +
|-
 +
! Gibbs Free Energy Change
 +
! Units
 +
! Species
 +
! Notes
 +
! Weight
 +
! Reference
 +
|-
 +
|(-69.979996)  
 +
|kcal/mol
 +
|Not stated
 +
|Estimated
 +
Enzyme: 15-LOX
 +
Substrate: Arachidonate
 +
Product: 15-HPETE
 +
pH: 7.3
 +
ionic strength: 0.25
 +
|64
 +
|<ref name="MetaCyc”>[http://metacyc.org/META/NEW-IMAGE?type=REACTION&object=RXN66-490 Caspi et al 2014, "The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases," Nucleic Acids Research 42:D459-D471]</ref>
 
|}
 
|}
 +
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 15-LOX Keq distribution
 +
! Mode !! Confidence Interval !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 2.27E+51 || 1.00E+01 || 1.19E+02 || 8.90E-01
 +
|}
 +
 +
[[Image:60.jpg|none|thumb|500px|The estimated probability distribution for 15-LOX Keq. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
  
 
== References ==
 
== References ==

Latest revision as of 09:19, 21 August 2019

Return to overview

The genes ALOX15B and ALOX15 encodes the formation of two isoforms of 15-lioxygenase (15-LOX). The gene ALOX15B encodes the formation of 15-LOX-2, which converts AA exclusively into 15(S)-HPETE. Whereas the gene ALOX15 encodes the 15-LOX-1 protein, which converts AA into both 12(S)-HPETE and 15(S)-HPETE. The formation of the unstable hydroperoxy fatty acids (HPETE) begins with the abstraction of a hydrogen radical at the allylic position between two double bonds. The structure undergoes a rearrangement reaction which results in the formation of a conjugated diene system. The insertion of molecular oxygen and a hydrogen leads to the formation of the final structure, a hydroperoxy fatty acid.

Reaction

R17 AA - HPETE15.jpg

Chemical equation

AA \rightleftharpoons 15-HPETE

Rate equation

R17.PNG

15-LOX Parameters

Kms

Literature values
Value Units Species Notes Weight Reference
3.70E-03 ± 3.00E-04 mM Human Expression Vector: Epithelium

Enzyme: 15-Lipoxygenase pH: 7.5 Temperature: 25

512 [1]
5.00E-03 mM Human Expression Vector: Reticulocyte

Enzyme: 15-Lipoxygenase pH:7.5 Temperature: Unspecified

512 [2]
1.06E-02 mM Human Expression Vector: Keratinocyte

Enzyme: 15-Lipoxygenase pH: 6.7 - 7.3 Temperature: 37

2048 [3]
1.17E-02 ± 9.00E-04 mM Human Expression Vector: Baculovirus Insect Cell

Enzyme: Wildtype 15-Lipoxygenase pH: 6.8 Temperature: 37

512 [4]
Description of the 15-LOX Kms distribution
Mode (mM) Confidence Interval Location parameter (µ) Scale parameter (σ)
1.02E-02 2.40E+00 -4.42E+00 4.10E-01
The estimated probability distribution for 15-LOX Kms. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.


Kmp

Description of the 15-LOX Kmp distribution
Mode (mM) Location parameter (µ) Scale parameter (σ)
1.02E-02 -4.42E+00 4.11E-01
The estimated probability distribution for 15-LOX Kmp. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

kcat

Literature values
Value Units Species Notes Weight Reference
595.8 ± 16.8 minute^{-1} Human Expression Vector: Epithelium

Enzyme: 15-Lipoxygenase pH: 7.5 Temperature: 25

512 [1]
37.2 ± 1.8 per minute Expression Vector:Human prostate epithelial

Enzyme:15-lipoxygenase-2

pH 7, Temperature: 22 512 [5]
45 ± 1.2 pH 7.5, Temperature: 22
44.4 ± 2.4 pH 8, Temperature: 22
34.2 ± 0.6 15°C, pH = 7.5
45 ± 1.2 22°C, pH = 7.5
62.4 ± 4.2 30°C, pH = 7.5
82.8 ± 4.8 37°C, pH = 7.5
Description of the 15-LOX kcat distribution
Mode (min-1) Confidence Interval Location parameter (µ) Scale parameter (σ)
6.51E+01 4.62E+00 4.60E+00 6.50E-01
The estimated probability distribution for 15-LOX kcat. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

Enzyme concentration

To convert the enzyme concentration from ppm to mM, the following equation was used.

Literature values
Value Units Species Notes Weight Reference
4.09 ppm Human Expression Vector: Lung

Enzyme: 15-LOX pH: 7.5 Temperature: 37 °C

1024 [6]
37.4 ppm Human Expression Vector: Spleen

Enzyme: 15-LOX pH: 7.5 Temperature: 37 °C

1024 [7]
1.40 ppm Human Expression Vector: Gut

Enzyme: 12-LOX pH: 7.5 Temperature: 37 °C

1024 [6]
Description of the 15-LOX concentration distribution
Mode (ppm) Mode (mM) Confidence Interval Location parameter (µ) Scale parameter (σ)
4.07 2.25E-05 7.23E+00 7.12E-01 1.19E+00
The estimated probability distribution for 15-LOX concentration. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

Keq

Literature values
Gibbs Free Energy Change Units Species Notes Weight Reference
(-69.979996) kcal/mol Not stated Estimated

Enzyme: 15-LOX Substrate: Arachidonate Product: 15-HPETE pH: 7.3 ionic strength: 0.25

64 [8]
Description of the 15-LOX Keq distribution
Mode Confidence Interval Location parameter (µ) Scale parameter (σ)
2.27E+51 1.00E+01 1.19E+02 8.90E-01
The estimated probability distribution for 15-LOX Keq. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

References

  1. ↑ 1.0 1.1 Jacquot C. "Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases. Biochemistry. 2008 Jul 8;47(27):7295-303. doi: 10.1021/bi800308q. Epub 2008 Jun 12.
  2. ↑ Jacquot C. "Synthesis of 11-Thialinoleic Acid and 14-Thialinoleic Acid, Inhibitors of Soybean and Human Lipoxygenases Org Biomol Chem. 2008 Nov 21; 6(22): 4242–4252.
  3. ↑ Burrall B. "Enzymatic properties of the 15-lipoxygenase of human cultured keratinocytes. J Invest Dermatol. 1988 Oct;91(4):294-7.
  4. ↑ Sloane D. L. "Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity. Protein Eng. 1995 Mar;8(3):275-82..
  5. ↑ Wecksler A. "Kinetic and Structural Investigations of the Allosteric Site in Human Epithelial 15-Lipoxygenase-2 Biochemistry, 2009, 48 (36), pp 8721–8730
  6. ↑ 6.0 6.1 M. Kim A draft map of the human proteome Nature, 2014 509, 575–581
  7. ↑ M. Wilhelm Mass-spectrometry-based draft of the human proteome Nature, 2014 509, 582–587
  8. ↑ Caspi et al 2014, "The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases," Nucleic Acids Research 42:D459-D471

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