Limonene-6-Hydroxylase (L6H)

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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 .

Standard Gibbs Free energy

Standard Gibbs free energy is -92.52051 kcal·mol^-1 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.
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.
val	µmol/min/mg (unit)	directionality	Organism	References

Kcat values

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

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.