Difference between revisions of "Limonene-6-Hydroxylase (L6H)"

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This enzyme is also known as (S)-limonene 6-monooxygenase, (-)-limonene 6-hydroxylase, (-)-limonene 6-monooxygenase, (-)-limonene,NADPH:oxygen oxidoreductase (6-hydroxylating)
 
This enzyme is also known as (S)-limonene 6-monooxygenase, (-)-limonene 6-hydroxylase, (-)-limonene 6-monooxygenase, (-)-limonene,NADPH:oxygen oxidoreductase (6-hydroxylating)
  
−
=== Issues ===
+
=== Reaction catalysed ===
−
 
 
−
 
 
−
=== Strategies ===
 
−
 
 
−
== Reaction catalysed ==
 
−
 
 
−
 
 
  
 
:<math>
 
:<math>
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</math>
 
</math>
  
−
== Metabolite and Enzyme Background Information ==
+
=== Metabolite and Enzyme Background Information ===
  
 
Long metabolite and enzyme names are abbreviated in the model for clarity and standard identification purposes.  
 
Long metabolite and enzyme names are abbreviated in the model for clarity and standard identification purposes.  
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| ChEMBL
 
| ChEMBL
 
| PubChem
 
| PubChem
−
| LIMONENE-6-MONOOXYGENASE-RXN
+
| [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=--LIMONENE-6-MONOOXYGENASE-RXN&redirect=T LIMONENE-6-MONOOXYGENASE-RXN]
 
|-
 
|-
 
| Metabolite
 
| Metabolite
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== Equation Rate ==
 
== Equation Rate ==
  
 +
This reaction is modelled using the reversible Michaelis-Menten equation, with two substrates; Limonene and NADPH, and two products; Carveol and NADP.
  
  
 
:<math>
 
:<math>
  
−
V_\mathrm{LimSynth} =  Vmax_\mathrm{forward} * \cfrac {\cfrac{[GPP]}{Km_\mathrm{GPP}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}}  + \cfrac {[PP]}{Km_\mathrm{PP}}  + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}
+
V_\mathrm{L6H} =  Vmax_\mathrm{forward} * \cfrac {\cfrac{[Limonene]}{Km_\mathrm{Limonene}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}}  + \cfrac {[PP]}{Km_\mathrm{PP}}  + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}
  
 
</math>
 
</math>
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{| class="wikitable"
 
{| class="wikitable"
−
! scope="col" style="width: 50px;" | Parameter
+
! scope="col" style="width: 50px;" | Parameter
 
! scope="col" style="width: 225px;" | Description  
 
! scope="col" style="width: 225px;" | Description  
−
! scope="col" style="width: 225px;" | Reference
+
! Units
 +
|-
 +
| V<sub>L6H</sub> || Net reaction rate for Limonene-6-Hydroxylase || Unit
 
|-
 
|-
−
| V<sub>LimSynth</sub> || Reaction rate for Limonene Synthase || ref
+
| V<sub>max<sub>forward</sub></sub> || Maximum reaction rate towards the production of trans-carveol || Unit
 
|-
 
|-
−
| V<sub>max<sub>forward</sub></sub> || Maximum reaction rate towards the production of limonene || ref
+
| Km<sub>limonene</sub> || Michaelis-Menten constant for Limonene || Unit
 
|-
 
|-
−
| Km<sub>GPP</sub> || Michaelis-Menten constant for GPP || ref
+
| Km<sub>carveol</sub> || Michaelis-Menten constant for trans-carveol || Unit
 
|-
 
|-
−
| Km<sub>Limonene</sub> || Michaelis-Menten constant for Limonene || ref
+
| Km<sub>NADPH</sub> || Michaelis-Menten constant for NADPH || Unit
 
|-
 
|-
−
| Km<sub>PP</sub> || Michaelis-Menten constant for PP || ref
+
| Km<sub>NADP</sub> || Michaelis-Menten constant for NADP || Unit
 
|-
 
|-
−
| K<sub>eq</sub> || Equilibrium constant || ref
+
| K<sub>eq</sub> || Equilibrium constant || Unit
 
|-
 
|-
−
| [GPP] || GPP concentration || ref
+
| [Limonene] || Limonene concentration || Unit
 
|-
 
|-
−
| [Limonene] || Limonene concentration || ref
+
| [Carveol] || trans-carveol concentration || Unit
 +
|-
 +
| [NADPH]|| NADPH concentration || Unit
 +
|-
 +
| [NADP]|| NADP concentration || Unit
 
|-
 
|-
−
| [PP]|| PP concentration || ref
 
 
|}
 
|}
  
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=== Calculating the Equilibrium Constant ===
 
=== Calculating the Equilibrium Constant ===
  
−
The equilibrium constant can be calculated using the Van't Hoff Isotherm equation:
+
The equilibrium constant can be calculated using the Van't Hoff Isotherm equation, which requires the information on the enzyme's standard Gibbs free energy <ref name="Liebermeister2005"> Liebermeister, W. & Klipp, E. 2005. http://pubman.mpdl.mpg.de/pubman/item/escidoc:1585440/component/escidoc:1585439/Liebermeister+et+al.+-+IEE+Proc.-Syst.+Biol.pdf."Biochemical networks with uncertain parameters." IEE Proc-Syst. Biol. 152(3): 97-105 </ref>.
 +
 
 +
==== Standard Gibbs Free energy ====
 +
 
 +
Standard Gibbs free energy is '''-92.52051 kcal·mol<sup>-1</sup> ''' <ref name= "Latendresse2013"> Latendresse, M. 2013. http://www.biocyc.org/PGDBConceptsGuide.shtml#gibbs. "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc." </ref>  according to MetaCyc [[http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=--LIMONENE-6-MONOOXYGENASE-RXN&redirect=T]].
 +
 
 +
SI derived unit for Gibbs free energy is Joules per mol (J mol<sup>-1</sup>). 1 kJ·mol<sup>−1</sup> is equal to 0.239 kcal·mol<sup>−1</sup>.
 +
 
 +
Therefore, the Gibbs free energy for L6H in kJ mol<sup>-1</sup> is:
 +
 
 +
:<math>
 +
 
 +
\cfrac {1}{0.239 kcal.mol^-1} * -92.52051 kcal.mol^-1
 +
 
 +
</math>
 +
 
 +
:<math>
 +
 
 +
= -387.115 kJmol^-1
 +
 
 +
</math>
 +
 
 +
==== The equilibrium constant ====
 +
 
 +
Using the Van't Hoff Isotherm equation:
 +
 
 
:<math>
 
:<math>
  
−
K_\mathrm{eq} = exp \left ( \cfrac {-?G^{°'}}{RT} \right )
+
K_\mathrm{eq} = exp \left ( \cfrac {-∆G^{°'}}{RT} \right )
  
 
</math>
 
</math>
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<math>
 
<math>
  
−
  = exp \left (  \cfrac {-(XY \text { kJmol}^{-1})}{ (8.31 \text{ JK}^{-1} \text { mol}^{-1} * 289 K} \right )
+
  = exp \left (  \cfrac {-(-387.1151 \text { kJmol}^{-1})}{ (8.31 \text{ JK}^{-1} \text { mol}^{-1} * 289 K} \right )
  
 
</math>
 
</math>
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<math>
 
<math>
  
−
  = exp \left (  \cfrac { XY \text { kJmol}^{-1} }{ 2401.59 \text{ JK}^{-1}\text { mol}^{-1} }\right)
+
  = exp \left (  \cfrac { 387.1151\text { kJmol}^{-1} }{ 2401.59 \text{ JK}^{-1}\text { mol}^{-1} }\right)
 
</math>
 
</math>
  
 
<math>
 
<math>
−
= exp \left ( \cfrac{ XY \text { Jmol}^{-1}}{2401.59 \text{ JK}^{-1}\text { mol}^{-1}} \right)
+
= exp \left ( \cfrac{ 387115.1 \text { Jmol}^{-1}}{2401.59 \text{ JK}^{-1}\text { mol}^{-1}} \right)
 
</math>
 
</math>
  
 
<math>
 
<math>
  
−
=exp \left ( XY \right )
+
=exp \left ( 161.19117 \right )
 
</math>
 
</math>
  
 
<math>
 
<math>
−
= (INSERT RESULT)
+
= 1.0103  \mathsf{x} 10^\mathsf{70}
 +
 
 +
 
 
</math>
 
</math>
  
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|-
 
|-
 
| style="width: 50pt;" | K<sub>eq</sub>
 
| style="width: 50pt;" | K<sub>eq</sub>
−
| style="width: 200pt;" | Equilibrium constant  
+
| Equilibrium constant  
  
 
|-
 
|-
−
| -?G<sup>°</sup> || Gibbs free energy change. For (INSERT ENZYME) it is (INSERT VALUE) kJmol<sup>-1</sup>
+
| -∆G<sup>°</sup> || Gibbs free energy change. For L6H it is 387.1151 kJmol<sup>-1</sup>
 
|-
 
|-
 
| R || Gas constant with a value of 8.31 JK<sup>-1</sup>mol<sup>-1</sup>
 
| R || Gas constant with a value of 8.31 JK<sup>-1</sup>mol<sup>-1</sup>
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| T || Temperature which is always expressed in kelvin
 
| T || Temperature which is always expressed in kelvin
 
|}
 
|}
−
 
−
==== Standard Gibbs Free energy ====
 
−
 
−
No information yet could be found on the standard Gibbs free energy for L6H. However, Metacyc has the standard Gibbs free energy value for Limonene oxide, catalysing the formation of cis-carveol from limonene [[http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-9465 RXN-9465]] as  '''-92.52051 kcal·mol<sup>-1</sup> ''' <ref name= "Latendresse2013"> Latendresse, M. 2013. http://www.biocyc.org/PGDBConceptsGuide.shtml#gibbs. "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc." </ref>.
 
  
 
=== Published Kinetic Parameter Values ===
 
=== Published Kinetic Parameter Values ===
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! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | References
 
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | References
 
|-
 
|-
−
| Value
+
| 20
−
| unit
+
| µM
−
| substrate
+
| Carveol
 +
| forward
 +
| Mentha sp.
 +
| <ref name="Karp1990">Karp, F. et. al. 1990. [http://www.ncbi.nlm.nih.gov/pubmed/2297225 "Monoterpene biosynthesis: Specificity of the hydroxylations of (-)-Limonene by enzyme preparations from Peppermint (Mentha piperita), Spearmint (Mentha spicata), and Perilla (Perilla frutescens) leaves", Archives of Biochemistry and Biophysics, 276(1):219-226]</ref>
 +
|-
 +
| value
 +
| µM
 +
| susbstrate
 
| directionality
 
| directionality
−
| organism  
+
| organism
−
| Ref
+
| ref
−
|-  
+
|-
 +
| value
 +
| µM
 +
| susbstrate
 +
| directionality
 +
| organism
 +
| ref
 +
|-
 +
| value
 +
| µM
 +
| susbstrate
 +
| directionality
 +
| organism
 +
| ref
 +
|-
 
|}  
 
|}  
  
−
[[File:LimSynth_01_Km.png|center|frame|μ :-5.05354 , σ :  0.49474]]
+
[[File:LimSynth_01_Km.png|center|frame|'''MOCK DISTRIBUTION''' μ :-5.05354 , σ :  0.49474]]
  
 
==== Vmax values ====
 
==== Vmax values ====
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! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | References
 
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | References
 
|-
 
|-
−
| Value
+
| 64
 +
| nmol/mg.h
 +
| forward
 +
| Mentha sp.
 +
| <ref name="Karp1990"></ref>
 +
|-
 +
| val
 
| µmol/min/mg (unit)
 
| µmol/min/mg (unit)
 
| directionality
 
| directionality

Latest revision as of 17:49, 17 March 2016

You can go back to main page of the kinetic model here.

What we know

This enzyme is also known as (S)-limonene 6-monooxygenase, (-)-limonene 6-hydroxylase, (-)-limonene 6-monooxygenase, (-)-limonene,NADPH:oxygen oxidoreductase (6-hydroxylating)

Reaction catalysed

Limonene + NADPH + O_2 \rightleftharpoons (-)-trans-carveol + NADP^+ + H_2O

Metabolite and Enzyme Background Information

Long metabolite and enzyme names are abbreviated in the model for clarity and standard identification purposes.

Metabolite Abbreviation Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem MetaCyc
(-)-4S-limonene Limonene C10H16 136.24 15384 449062 22311 or 439250
(-)-trans-carveol carveol Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem MetaCyc
NADPH NADPH Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem MetaCyc
NADP NADP Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem MetaCyc
Limonene-6-hydroxylase L6H Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem LIMONENE-6-MONOOXYGENASE-RXN
Metabolite Abbreviation Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem MetaCyc

Equation Rate

This reaction is modelled using the reversible Michaelis-Menten equation, with two substrates; Limonene and NADPH, and two products; Carveol and NADP.


V_\mathrm{L6H} = Vmax_\mathrm{forward} * \cfrac {\cfrac{[Limonene]}{Km_\mathrm{Limonene}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}} + \cfrac {[PP]}{Km_\mathrm{PP}} + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}


Parameter Description Units
VL6H Net reaction rate for Limonene-6-Hydroxylase Unit
Vmaxforward Maximum reaction rate towards the production of trans-carveol Unit
Kmlimonene Michaelis-Menten constant for Limonene Unit
Kmcarveol Michaelis-Menten constant for trans-carveol Unit
KmNADPH Michaelis-Menten constant for NADPH Unit
KmNADP Michaelis-Menten constant for NADP Unit
Keq Equilibrium constant Unit
[Limonene] Limonene concentration Unit
[Carveol] trans-carveol concentration Unit
[NADPH] NADPH concentration Unit
[NADP] NADP concentration Unit

Strategies for estimating the kinetic parameter values

Calculating the Equilibrium Constant

The equilibrium constant can be calculated using the Van't Hoff Isotherm equation, which requires the information on the enzyme's standard Gibbs free energy [1].

Standard Gibbs Free energy

Standard Gibbs free energy is -92.52051 kcal·mol-1 [2] according to MetaCyc [[1]].

SI derived unit for Gibbs free energy is Joules per mol (J mol-1). 1 kJ·mol−1 is equal to 0.239 kcal·mol−1.

Therefore, the Gibbs free energy for L6H in kJ mol-1 is:

\cfrac {1}{0.239 kcal.mol^-1} * -92.52051 kcal.mol^-1
= -387.115 kJmol^-1

The equilibrium constant

Using the Van't Hoff Isotherm equation:

K_\mathrm{eq} = exp \left ( \cfrac {-∆G^{°'}}{RT} \right )


= exp \left ( \cfrac {-(-387.1151 \text { kJmol}^{-1})}{ (8.31 \text{ JK}^{-1} \text { mol}^{-1} * 289 K} \right )

= exp \left ( \cfrac { 387.1151\text { kJmol}^{-1} }{ 2401.59 \text{ JK}^{-1}\text { mol}^{-1} }\right)

= exp \left ( \cfrac{ 387115.1 \text { Jmol}^{-1}}{2401.59 \text{ JK}^{-1}\text { mol}^{-1}} \right)

=exp \left ( 161.19117 \right )

= 1.0103 \mathsf{x} 10^\mathsf{70}


where;

Keq Equilibrium constant
-∆G° Gibbs free energy change. For L6H it is 387.1151 kJmol-1
R Gas constant with a value of 8.31 JK-1mol-1
T Temperature which is always expressed in kelvin

Published Kinetic Parameter Values

Km Values

Km (mM) Unit Substrate / Product Directionality Organism References
20 µM Carveol forward Mentha sp. [3]
value µM susbstrate directionality organism ref
value µM susbstrate directionality organism ref
value µM susbstrate directionality organism ref
MOCK DISTRIBUTION μ :-5.05354 , σ : 0.49474

Vmax values

Vmax Unit Directionality Organism References
64 nmol/mg.h forward Mentha sp. [3]
val µmol/min/mg (unit) directionality Organism References

Kcat values

Kcat Unit Organism Reference
value s-1 Organism ref e.g. Alonso 1992 [4]

Extracting Information from (INSERT SUBSTRATE/PRODUCT) Production Rates

Amount produced (mg/L) Time (H) Organism Description Reaction Flux (µM/s)
X X Y Z Z
X X Y Z Z
X X Y Z Z
X X Y Z Z
X X Y Z Z
X X Y Z Z

Published Kinetic Parameter Values

Km (mM) Vmax Kcat (s-1) Kcat/Km Organism Description
0.00125 - - Z A -> B
0.0018 - - - Z A -> B
Y Y Y Y Z A -> B
Y Y Y Y Z A -> B
Y Y - - Z A -> B
Y - Y - Z GPP -> B
Y - Y - Z GPP -> B
x - y - Z. A -> B

Detailed descriptions of kinetic values used in this model

A more detailed description of the kinetic values listed above can be found here.

Simulations

References

  1. ↑ Liebermeister, W. & Klipp, E. 2005. http://pubman.mpdl.mpg.de/pubman/item/escidoc:1585440/component/escidoc:1585439/Liebermeister+et+al.+-+IEE+Proc.-Syst.+Biol.pdf."Biochemical networks with uncertain parameters." IEE Proc-Syst. Biol. 152(3): 97-105
  2. ↑ Latendresse, M. 2013. http://www.biocyc.org/PGDBConceptsGuide.shtml#gibbs. "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."
  3. ↑ 3.0 3.1 Karp, F. et. al. 1990. "Monoterpene biosynthesis: Specificity of the hydroxylations of (-)-Limonene by enzyme preparations from Peppermint (Mentha piperita), Spearmint (Mentha spicata), and Perilla (Perilla frutescens) leaves", Archives of Biochemistry and Biophysics, 276(1):219-226
  4. ↑ Alonso et. al. 1992. "Purification of 4S-Limonene Synthase, a Monoterpene Cyclase from the Glandular Trichomes of Peppermint (Mentha x piperita) and Spearmint (Mentha spicata)", The Journal of Biological Chemistry, 267(11):7582-7587
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