Double-bond reductase (DBR)

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What we know

Pulegone reductase(s) (PGR) catalyses the NADPH-dependent convertion of pulegone to menthone and isomenthone (the former predominates).

Reaction catalysed

$pulegone + NADPH \rightleftharpoons menthone + NADP^+$

$pulegone + NADPH \rightleftharpoons isomenthone + NADP^+$

Enzyme and Metabolite Background Information

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

Metabolite	Abbreviation	Chemical Formula	Molar mass (g/mol)	ChEBI	ChEMBL	PubChem	BRENDA	PlantCyc
pulegone reductase	PGR		37914 Da				1.3.1.81
pulegone		C10H16O	136.24
menthone
NADPH		C21H30N7O17P3	745.42116	16474
NADP+		C21H29N7O17P3	744.41322	18009
isomenthone

Equation Rate

Two PGR reactions are included in the kinetic model with each converting pulegone to methone and isomenthone respectively. Both reactions are parameterised using random Bi-Bi reversible Michaelis-Menten equation.

Reaction 1: Conversion of pulegone to menthone

Failed to parse (Cannot store math image on filesystem.): V_\mathrm{PGR} = Kcat_\mathrm{forward} * [PGR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[menthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )} { \left (1 + \cfrac {[NADPH]}{Km_\mathrm{NADPH}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right ) + \left ( 1+ \cfrac {[pulegone]}{Km_\mathrm{pulegone}} + \cfrac {[menthone]}{Km_\mathrm{menthone}} \right ) }

Reaction 2: Conversion of pulegone to isomenthone

Failed to parse (Cannot store math image on filesystem.): V_\mathrm{PGR} = Kcat_\mathrm{forward} * [PGR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[isomenthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )} { \left (1 + \cfrac {[NADPH]}{Km_\mathrm{NADPH}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right ) + \left ( 1+ \cfrac {[pulegone]}{Km_\mathrm{pulegone}} + \cfrac {[isomenthone]}{Km_\mathrm{isomenthone}} \right ) }

Parameter	Description	Units
VPGR	Reaction rate for Limonene-3-hydroxylase	μM/min
Kcatforward	Catalytic constant in the forward direction	s-1
Kmpulegone	Michaelis-Menten constant for pulegone	μM
Kmmenthone	Michaelis-Menten constant for menthone	μM
Kmisomenthone	Michaelis-Menten constant for isomenthone	μM
KmNADPH	Michaelis-Menten constant for NADPH	μM
KmNADP	Michaelis-Menten constant for NADP+	μM
Keq	Equilibrium constant
[PGR]	enzyme concentration	μM
[pulegone]	Pulegone concentration	μM
[menthone]	Menthone concentration	μM
[isomenthone]	Isomenthone concentration	μM
[NADPH]	NADPH concentration	μM
[NADP]	NADP+ concentration	μM

Strategies for estimating the kinetic parameter values

Standard Gibbs Free energy

The Gibbs free energy for PGR is -3.9565125 kcal.mol^-1. This value is estimated from the 'Contribution group' method by Latendresse, M. and is available from MetaCyc (EC 1.3.1.81) .

Calculating the Equilibrium Constant

The equilibrium constant can be calculated using the Van't Hoff Isotherm equation:

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

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

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

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

$=exp \left ( XY \right )$

$= (INSERT RESULT)$

where;

Keq	Equilibrium constant
-ΔG°	Gibbs free energy change. For (INSERT ENZYME) it is (INSERT VALUE) kJmol-1
R	Gas constant with a value of 8.31 JK-1mol-1
T	Temperature which is always expressed in kelvin

Extracting Information from menthone Production Rates

A table will go here

Published Kinetic Parameter Values

Km Values

Parameter	Directionality	Substrate / Product	Value	unit	Method notes	References
Km	Forward	pulegone	2.3	µM	Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45	Ringer2003
Km	Forward	pulegone	2.9	µM	Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45, Km 2.3 +/- 0.6	Ringer2003
Km	Forward	NADPH	6.9	µM	Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45	Ringer2003

Detailed description of kinetic values obtained from literature

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