Difference between revisions of "Transformation of AA to 12-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 12- LOX enzyme catalyses the addition of O2 at the C-12 position of AA, producing 12-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 ==
  
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==Chemical equation==
 
==Chemical equation==
  
−
<center><math> AA \rightleftharpoons PGH2 </math></center>
+
<center><math> AA \rightleftharpoons 12-HPETE </math></center>
  
 
== Rate equation ==
 
== Rate equation ==
  
 +
[[File:R19.PNG|center|500px]]
  
−
== 12-LOX Parameters ==
+
== Enzyme 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 24:
 
! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
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|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Platelet
+
|Expression Vector: Platelet
 +
Enzyme: 12-Lipoxygenase
 +
pH: 7.4
 +
Temperature: 37°C.
 +
|2048
 
|<ref name="Lagarde1984"> [http://www.ncbi.nlm.nih.gov/pubmed/6433902 Lagarde M. "Subcellular localization and some properties of lipoxygenase activity in human blood platelets.'' Biochem J. 1984 Sep 1;222(2):495-500.]</ref>   
 
|<ref name="Lagarde1984"> [http://www.ncbi.nlm.nih.gov/pubmed/6433902 Lagarde M. "Subcellular localization and some properties of lipoxygenase activity in human blood platelets.'' Biochem J. 1984 Sep 1;222(2):495-500.]</ref>   
 
|-
 
|-
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|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Platelet
+
|Expression Vector: Platelet
 +
Enzyme: 12-Lipoxygenase
 +
pH: 7
 +
Temperature: 24
 +
|512
 
|<ref name="Hada1991"> [http://www.ncbi.nlm.nih.gov/pubmed/1851637 Hada T. "Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins.'' Biochim Biophys Acta. 1991 Apr 24;1083(1):89-93.]</ref>   
 
|<ref name="Hada1991"> [http://www.ncbi.nlm.nih.gov/pubmed/1851637 Hada T. "Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins.'' Biochim Biophys Acta. 1991 Apr 24;1083(1):89-93.]</ref>   
 
|-
 
|-
 
|1.00E-02
 
|1.00E-02
 
|<math> mM </math>
 
|<math> mM </math>
−
|Human
+
|Human Platlet
−
|Recombinant Histidine-Tagged Human Platelet 12-Lipoxygenase
+
|Expression Vector: Baculovirus
 +
Enzyme: 12-Lipoxygenase
 +
pH: 8
 +
Temperature: 37
 +
|256
 
|<ref name="Chen1993"> [http://www.ncbi.nlm.nih.gov/pubmed/8319693 Chen X. S. "Purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system.'' Eur J Biochem. 1993 Jun 15;214(3):845-52.]</ref>   
 
|<ref name="Chen1993"> [http://www.ncbi.nlm.nih.gov/pubmed/8319693 Chen X. S. "Purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system.'' Eur J Biochem. 1993 Jun 15;214(3):845-52.]</ref>   
 
|-
 
|-
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|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Platelet 12-Lipoxygenase
+
|Expression Vector:  Platelets
 +
Enzyme:12-Lipoxygenase
 +
pH: 7.4
 +
Temperature: 37
 +
|2048
 
|<ref name="Romano1993"> [http://www.ncbi.nlm.nih.gov/pubmed/8250832 Romano M. "Lipoxin synthase activity of human platelet 12-lipoxygenase.'' Biochem J. 1993 Nov 15;296 ( Pt 1):127-33.]</ref>   
 
|<ref name="Romano1993"> [http://www.ncbi.nlm.nih.gov/pubmed/8250832 Romano M. "Lipoxin synthase activity of human platelet 12-lipoxygenase.'' Biochem J. 1993 Nov 15;296 ( Pt 1):127-33.]</ref>   
 
|-
 
|-
 
|}
 
|}
  
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 12-LOX Kms distribution
 +
! Mode (mM) !! Confidence Interval !! Location parameter (σ) !! Scale parameter (σ)
 +
|-
 +
| 7.60E-03 || 4.34E+00 || -4.48E+00 || 6.30E-01
 +
|}
 +
 +
[[Image:61.jpg|none|thumb|500px|The estimated probability distribution for 12-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>===
 +
This is a “Dependent parameter”, meaning that the log-normal distribution for this parameter was calculated using multivariate distributions  (this is discussed in detail[[Quantification of parameter uncertainty | here]]). As a result, no confidence interval factor or literature values were cited for this parameter.
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 12-LOX Kmp distribution
 +
! Mode (mM) !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 7.30E-03 || -4.52E+00 || 6.28E-01
 +
|}
 +
 +
[[Image:62.jpg|none|thumb|500px|The estimated probability distribution for 12-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
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! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
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|per minute
 
|per minute
 
|Human
 
|Human
−
|Human reticulocyte 15-lipoxygenase-1, Enzymatic assays were performed in 25 mM Hepes buffer (pH 7.5,
+
|Expression Vector: Human reticulocyte
−
25C), with and without product (12-HETE) addition, in the presence of 25 μM of substrate.
+
Enzyme: 15-lipoxygenase-1
 +
pH: 7.5
 +
Temperature: 25
 +
|256
 
|<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>
 
|<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>
 
|-
 
|-
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|per minute
 
|per minute
 
|Wild Boar
 
|Wild Boar
−
|Wild Type Leukocyte-Type 12-Lipoxygenase Expressed in E. coli.
+
|Expression Vector: E. coli.
 +
Enzyme: 12-Lipoxygenase
 +
pH: 7.4
 +
Temperature: 37
 +
|1024
 
|<ref name="Richards1997"> [http://pubs.acs.org/doi/abs/10.1021/bi963051a Richards K. "Leukocyte 12-Lipoxygenase:  Expression, Purification, and Investigation of the Role of Methionine Residues in Turnover-Dependent Inactivation and 5,8,11,14-Eicosatetraynoic Acid Inhibition'' Biochemistry, 1997, 36 (22), pp 6692–6699]</ref>
 
|<ref name="Richards1997"> [http://pubs.acs.org/doi/abs/10.1021/bi963051a Richards K. "Leukocyte 12-Lipoxygenase:  Expression, Purification, and Investigation of the Role of Methionine Residues in Turnover-Dependent Inactivation and 5,8,11,14-Eicosatetraynoic Acid Inhibition'' Biochemistry, 1997, 36 (22), pp 6692–6699]</ref>
 
|-
 
|-
 
|}
 
|}
 +
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the 12-LOX kcat distribution
 +
! Mode (min-1) !! Confidence Interval !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 4.87E+02 || 1.20E+00 || 6.22E+00 || 1.80E-01
 +
|}
 +
 +
[[Image:63.jpg|none|thumb|500px|The estimated probability distribution for 12-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;" | 12-LOX Abundance
 
|-
 
|-
 
! Value
 
! Value
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! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 +
! Reference
 +
|-
 +
|19.8
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Spleen
 +
Enzyme: 12-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>
 +
|-
 +
|1.60
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Liver
 +
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>
 +
|-
 +
|0.28
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Gut
 +
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>
 +
|-
 +
|0.11
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Pancreas
 +
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 12-LOX concentration distribution
 +
! Mode (ppm) !! Mode (mM) !! Confidence Interval !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 4.98E-01 ||2.76E-06 || 7.23E+00 || 7.12E-01 || 1.19E+00
 +
|}
 +
 +
[[Image:156.jpg|none|thumb|500px|The estimated probability distribution for 12-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
 
! Reference
 
|-
 
|-
−
|5.00e-06 - 6.00e-05
+
|(-69.979996)
−
|mM
+
|kcal/mol
−
|Human
+
|Not stated
−
|Human reticulocyte 15-lipoxygenase-1, Enzymatic assays were performed in 25 mM Hepes buffer (pH 7.5,
+
|Estimated
−
25C), with and without product (12-HETE) addition, in the presence of 25 μM of substrate.
+
Enzyme: 12-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>
+
Substrate: Arachidonate
 +
Product: 12-HPETE
 +
pH: 7.3
 +
ionic strength: 0.25
 +
|64
 +
|<ref name="MetaCyc”>[http://metacyc.org/META/NEW-IMAGE?type=REACTION&object=ARACHIDONATE-12-LIPOXYGENASE-RXN 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 12-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:64.jpg|none|thumb|500px|The estimated probability distribution for 12-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 08:18, 21 August 2019

Return to overview

The 12- LOX enzyme catalyses the addition of O2 at the C-12 position of AA, producing 12-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

R179AA - HPETE12.jpg

Chemical equation

AA \rightleftharpoons 12-HPETE

Rate equation

R19.PNG

Enzyme Parameters

Kms

Literature values
Value Units Species Notes Weight Reference
7.20E-03 mM Human Expression Vector: Platelet

Enzyme: 12-Lipoxygenase pH: 7.4 Temperature: 37°C.

2048 [1]
8.00E-02 mM Human Expression Vector: Platelet

Enzyme: 12-Lipoxygenase pH: 7 Temperature: 24

512 [2]
1.00E-02 mM Human Platlet Expression Vector: Baculovirus

Enzyme: 12-Lipoxygenase pH: 8 Temperature: 37

256 [3]
7.90E-03 ± 8.00E-04 mM Human Expression Vector: Platelets

Enzyme:12-Lipoxygenase pH: 7.4 Temperature: 37

2048 [4]
Description of the 12-LOX Kms distribution
Mode (mM) Confidence Interval Location parameter (σ) Scale parameter (σ)
7.60E-03 4.34E+00 -4.48E+00 6.30E-01
The estimated probability distribution for 12-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

This is a “Dependent parameter”, meaning that the log-normal distribution for this parameter was calculated using multivariate distributions (this is discussed in detail here). As a result, no confidence interval factor or literature values were cited for this parameter.

Description of the 12-LOX Kmp distribution
Mode (mM) Location parameter (µ) Scale parameter (σ)
7.30E-03 -4.52E+00 6.28E-01
The estimated probability distribution for 12-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
336 ± 12 per minute Human Expression Vector: Human reticulocyte

Enzyme: 15-lipoxygenase-1 pH: 7.5 Temperature: 25

256 [5]
504 per minute Wild Boar Expression Vector: E. coli.

Enzyme: 12-Lipoxygenase pH: 7.4 Temperature: 37

1024 [6]
Description of the 12-LOX kcat distribution
Mode (min-1) Confidence Interval Location parameter (µ) Scale parameter (σ)
4.87E+02 1.20E+00 6.22E+00 1.80E-01
The estimated probability distribution for 12-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.

12-LOX Abundance
Value Units Species Notes Weight Reference
19.8 ppm Human Expression Vector: Spleen

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

1024 [7]
1.60 ppm Human Expression Vector: Liver

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

1024 [8]
0.28 ppm Human Expression Vector: Gut

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

1024 [8]
0.11 ppm Human Expression Vector: Pancreas

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

1024 [8]
Description of the 12-LOX concentration distribution
Mode (ppm) Mode (mM) Confidence Interval Location parameter (µ) Scale parameter (σ)
4.98E-01 2.76E-06 7.23E+00 7.12E-01 1.19E+00
The estimated probability distribution for 12-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: 12-LOX Substrate: Arachidonate Product: 12-HPETE pH: 7.3 ionic strength: 0.25

64 [9]
Description of the 12-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 12-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. ↑ Lagarde M. "Subcellular localization and some properties of lipoxygenase activity in human blood platelets. Biochem J. 1984 Sep 1;222(2):495-500.
  2. ↑ Hada T. "Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins. Biochim Biophys Acta. 1991 Apr 24;1083(1):89-93.
  3. ↑ Chen X. S. "Purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system. Eur J Biochem. 1993 Jun 15;214(3):845-52.
  4. ↑ Romano M. "Lipoxin synthase activity of human platelet 12-lipoxygenase. Biochem J. 1993 Nov 15;296 ( Pt 1):127-33.
  5. ↑ [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]
  6. ↑ Richards K. "Leukocyte 12-Lipoxygenase:  Expression, Purification, and Investigation of the Role of Methionine Residues in Turnover-Dependent Inactivation and 5,8,11,14-Eicosatetraynoic Acid Inhibition Biochemistry, 1997, 36 (22), pp 6692–6699
  7. ↑ M. Wilhelm Mass-spectrometry-based draft of the human proteome Nature, 2014 509, 582–587
  8. ↑ 8.0 8.1 8.2 M. Kim A draft map of the human proteome Nature, 2014 509, 575–581
  9. ↑ 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

Related Reactions

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