Text S1: Model description - PLOS

Text S1: Model description

1 Ordinary differential equations Based on the reaction scheme in Figure 1, a set of ordinary differential equations was constructed. As indicated in the figure, most of the enzymes catalyze multiple reactions, i.e. with substrates of different chain length, and many substrates can be converted by different enzymes. For instance vcpt1C16 is the rate of conversion of C16 (palmitoyl) CoA by CPT1. In the abbreviations CYT indicates the cytosolic metabolite pool and MAT the metabolite pool in the mitochondrial matrix. 𝑑C16AcylCarCYT 𝑑t

𝑑C16AcylCarMAT 𝑑t

=

𝑑C16AcylCoAMAT 𝑑t

𝑣cpt1C16 −𝑣cactC16

(1)

𝑣cactC16 −𝑣cpt2C16

(2)

𝑉CYT

=

=

𝑑C16EnoylCoAMAT 𝑑t

𝑉MAT

𝑣cpt2C16 −𝑣vlcadC16 −𝑣lcadC16

=

𝑉MAT

𝑣vlcadC16 +𝑣lcadC16 −𝑣crotC16 −𝑣mtpC16

𝑑C16HydroxyacylCoAMAT 𝑑t 𝑑C16KetoacylCoAMAT 𝑑t 𝑑C14AcylCarCYT 𝑑t


=


=

𝑉MAT

=



=

𝑉MAT

𝑉MAT

𝑣crotC14 −𝑣mschadC14 𝑉MAT

𝑣mschadC14 −𝑣mckatC14 𝑉MAT

−𝑣cactC12 𝑉CYT

=


=

𝑉MAT

𝑣vlcadC12 +𝑣lcadC12 +𝑣mcadC12 −𝑣crotC12 −𝑣mtpC12

𝑑C12HydroxyacylCoAMAT 𝑑t

=

(9) (10) (11) (12)

(14)

𝑣cpt2C12 +𝑣mtpC14 +𝑣mckatC14 −𝑣vlcadC12 −𝑣lcadC12 −𝑣mcadC12

=

(6)

(13)

𝑣cactC12 −𝑣cpt2C12 𝑉MAT

(5)

(8)

𝑣vlcadC14 +𝑣lcadC14 −𝑣crotC14 −𝑣mtpC14

=

(4)

(7)

𝑣cpt2C14 +𝑣mtpC16 +𝑣mckatC16 −𝑣vlcadC14 −𝑣lcadC14

𝑑C14KetoacylCoAMAT 𝑑t

=



𝑑C14HydroxyacylCoAMAT 𝑑t 𝑑C12AcylCarCYT 𝑑t



=


=

=

𝑉MAT

(3)

𝑉MAT


(15) (16) (17)

𝑑C12KetoacylCoAMAT 𝑑t 𝑑C10AcylCarCYT 𝑑t


=


=



=



=

𝑣cpt2C10 +𝑣mtpC12 +𝑣mckatC12 −𝑣lcadC10 −𝑣mcadC10

𝑑C10KetoacylCoAMAT 𝑑t 𝑑C8AcylCarCYT 𝑑t


=


=

=


=

=



=




=


𝑉MAT

𝑣mcadC6 +𝑣scadC6 −𝑣crotC6 𝑉MAT

=

𝑣cactC4 −𝑣cpt2C4 𝑉MAT

𝑣cpt2C4 +𝑣mckatC6 −𝑣mcadC4 −𝑣scadC4

=

𝑉MAT

𝑣mcadC4 +𝑣scadC4 −𝑣crotC4 𝑉MAT

𝑑C4HydroxyacylCoAMAT 𝑑t 𝑑C4AcetoacetylCoAMAT 𝑑t

=

=

(22) (23) (24) (25)

(27) (28) (29) (30) (31) (32)

𝑣cpt2C6 +𝑣mtpC8 +𝑣mckatC8 −𝑣mcadC6 −𝑣scadC6

=

=


𝑉MAT

𝑑C6KetoacylCoAMAT 𝑑t

=




𝑉MAT


=


𝑉MAT

𝑣lcadC8 +𝑣mcadC8 −𝑣crotC8 −𝑣mtpC8

=

(21)

(26)

𝑣cpt2C8 +𝑣mtpC10 +𝑣mckatC10 −𝑣lcadC8 −𝑣mcadC8

𝑑C8KetoacylCoAMAT 𝑑t 𝑑C6AcylCarMAT 𝑑t


𝑉MAT

=

=




=

𝑉MAT


=


𝑉MAT

𝑣lcadC10 +𝑣mcadC10 −𝑣crotC10 −𝑣mtpC10

𝑑C10HydroxyacylCoAMAT 𝑑t

(19) (20)

𝑉MAT

=

(18)



(33) (34) (35) (36) (37) (38) (39) (40) (41) (42)

𝑑AcetylCoAMAT 𝑑t 𝑑FADHMAT 𝑑t

𝑑NADHMAT 𝑑t

=

=

=

𝑣mtpC16 +𝑣mckatC16 +𝑣mtpC14 +𝑣mckatC14 +𝑣mtpC12 +𝑣mckatC12 +𝑣mtpC10 +𝑣mckatC10 +𝑣mtpC8 +𝑣mckatC8 +𝑣mckatC6 +2 ∙ 𝑣mckatC4 −𝑣acesink 𝑉MAT

𝑣vlcadC16 +𝑣vlcadC14 +𝑣vlcadC12 +𝑣lcadC16 +𝑣lcadC14 +𝑣lcadC12 +𝑣lcadC10 +𝑣lcadC8 +𝑣mcadC12 +𝑣mcadC10 +𝑣mcadC8 +𝑣mcadC6 +𝑣mcadC4 +𝑣scadC6 +𝑣scadC4 −𝑣fadhsink 𝑉MAT

𝑣mtpC16 +𝑣mtpC14 +𝑣mtpC12 +𝑣mtpC10 +𝑣mtpC8 +𝑣mschadC16 +𝑣mschadC14 +𝑣mschadC12 +𝑣mschadC10 +𝑣mschadC8 +𝑣mschadC6 +𝑣mschadC4 −𝑣nadhsink 𝑉MAT

(43)

(44) (45)

2 Kinetic rate equations When an enzyme catalyzes the conversion of multiple substrates, the same rate equation applies, but many of the rate constants are chain-length-specific, as indicated by the subscript n. Most equations are of the reversible Michaelis-Menten type (based on random binding of substrates). The only exception is the rate equations for the transporter CACT. In the model description the rates are given in µmol.min-1.mgProtein-1, while the rates of change in the differential equations are in µM.min-1. In the presentation of the results the fluxes were converted to µmol.min-1.gProtein-1. As in the Modre-Osprian model (1), the rate equations for the consumption of the end products NADH, FADH2 and acetyl CoA were made up such that i) the sink reactions do not control the flux; and ii) the concentrations of these metabolites equal the constant K1xsink. For the computational outcome this is equivalent to fixing the concentrations of NADH, FADH2 and acetyl CoA as external parameters. The advantage of the formulation used here, is that it allows to directly monitor the fluxes of end product removal. 𝑣cpt1C16 =

C16AcylCoACYT∙CarCYT C16AcylCarCYT[t]∙CoACYT − � 𝐾𝑚C16AcylCoACYT ∙𝐾𝑚CarCYT 𝐾𝑚C16AcylCoACYT ∙𝐾𝑚CarCYT ∙𝐾𝑒𝑞cpt1 𝑛cpt1 C16AcylCoACYT C16AcylCarCYT[t] MalCoACYT CarCYT CoACYT + +� � + � �∙�1+𝐾𝑚 �1+𝐾𝑚 𝐾𝑖MalCoACYT CarCYT 𝐾𝑚CoACYT C16AcylCoACYT 𝐾𝑚C16AcylCarCYT

𝑠𝑓cpt1C16 ∙𝑉cpt1 ∙�

𝑣cactCn (n → 4, 6, 8, 10, 12, 14 𝑜𝑟 16) =

𝑣cpt2Cn (n → 4, 6, 8, 10, 12, 14 𝑜𝑟 16) = 𝑣vlcadCn (n → 12, 14 𝑜𝑟 16) =

(46) CnAcylCarMAT[t]∙CarCYT � 𝐾𝑒𝑞cact

𝑉𝑓cact ∙�CnAcylCarCYT[t]∙CarMAT−

+

CnAcylCarCYT[t]∙CarMAT+𝐾𝑚CarMAT ∙CnAcylCarCYT[t]+𝐾𝑚CnAcylCarCYT ∙CarMAT∙�1+

CarCYT � 𝐾𝑖CatCYT

(47)

𝑉𝑓cact CnAcylCarCYT[t] ∙�𝐾𝑚CarCYT ∙CnAcylCarMAT[t]∙�1+ �+CarCYT∙�𝐾𝑚CnAcylCarMAT[t] +CncylCarMAT[t]�� 𝑉𝑟cact ∙𝐾𝑒𝑞cact 𝐾𝑖CnAcylCarCYT[t] CnAcylCarMAT[t]∙CoAMAT CnAcylCoAMAT[t]∙CarMAT − � 𝐾𝑚CnAcylCarMAT[t] ∙𝐾𝑚CoAMAT 𝐾𝑚CnAcylCarMAT[t] ∙𝐾𝑚CoAMAT ∙𝐾𝑒𝑞cpt2

𝑠𝑓cpt2Cn ∙𝑉cpt2 ∙�

CnAcylCarMAT[t]

CnAcylCoAMAT[t] CoAMAT CarMAT + � ��∙�1+𝐾𝑚 CoAMAT 𝐾𝑚CarMAT CnAcylCarMAT[t] 𝐾𝑚CnAcylCoAMAT[t]

�1+∑Cn n→4,6,8,10,12,14 𝑎𝑛𝑑 16�𝐾𝑚

CnAcylCoAMAT[t]∙�FADtMAT-FADHMAT[t]� CnEnoylCoAMAT[t]∙FADHMAT[t] − � 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT ∙𝐾𝑒𝑞vlcad

𝑠𝑓vlcadCn ∙𝑉vlcad ∙�

CnAcylCoAMAT[t]

CnEnoylCoAMAT[t] FADtMAT-FADHMAT[t] FADHMAT[t] + � ��∙�1+ 𝐾𝑚FADMAT 𝐾𝑚FADHMAT CnAcylCoAMAT[t] 𝐾𝑚CnEnoylCoAMAT[t]

�1+∑Cn n→12,14 𝑎𝑛𝑑 16)�𝐾𝑚

+

(48)

+

(49)

𝑣lcadCn (n → 8, 10, 12, 14 𝑜𝑟 16) = 𝑣mcadCn (n → 4, 6, 8, 10 𝑜𝑟 12) = 𝑣scadCn (n → 4 𝑜𝑟 6) =

CnAcylCoAMAT[t]∙�FADtMAT-FADHMAT[t]� CnEnoylCoAMAT[t]∙FADHMAT[t] − � 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT ∙𝐾𝑒𝑞lcad

𝑠𝑓lcadCn ∙𝑉lcad ∙�

CnAcylCoAMAT[t]


�1+∑Cn n→8,10,12,14 𝑎𝑛𝑑 16)�𝐾𝑚

CnAcylCoAMAT[t]∙�FADtMAT-FADHMAT[t]� CnEnoylCoAMAT[t]∙FADHMAT[t] − � 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT ∙𝐾𝑒𝑞mcad

𝑠𝑓mcadCn ∙𝑉mcad ∙�

CnAcylCoAMAT[t]


�1+∑Cn n→4,6,8,10 𝑎𝑛𝑑 12�𝐾𝑚

CnAcylCoAMAT[t]


�1+∑Cn n→4 𝑎𝑛𝑑 6�𝐾𝑚

𝑣crotCn (n → 4, 6, 8, 10, 12, 14 𝑜𝑟 16) =

CnEnoylCoAMAT[t] CnHydroxyacylCoAMAT[t] AcetoacetylCoAMAT[t] + �+𝐾𝑖 𝐾𝑚CnEnoylCoAMAT[t] 𝐾𝑚CnHydroxyacylCoAMAT[t] AcetoacetylCoAMAT

1+∑Cn n→4,6,8,10,12,14 𝑎𝑛𝑑 16�

𝑣mckatCn (n → 4, 6, 8, 10, 12, 14 𝑜𝑟 16) =

CnEnoylCoAMAT[t] CnHydroxyacylCoAMAT[t] − � 𝐾𝑚CnEnoylCoAMAT[t] 𝐾𝑚CnEnoylCoAMAT[t] ∙𝐾𝑒𝑞crot

𝑠𝑓crotCn ∙𝑉crot ∙�

𝑣mschadCn (n → 4, 6, 8, 10, 12, 14 𝑜𝑟 16) =

𝑣mtpCn (n → 8, 10, 12, 14 𝑜𝑟 16) =

+

CnHydroxyacylCoAMAT[t]∙�NADtMAT-NADHMAT[t]� CnketoacylCoAMAT[t]∙NADHMAT[t] − � 𝐾𝑚CnHydroxyacylCoAMAT[t] ∙𝐾𝑚NADMAT 𝐾𝑚CnHydroxyacylCoAMAT[t] ∙𝐾𝑚NADMAT ∙𝐾𝑒𝑞mschad

𝑠𝑓mschadCn ∙𝑉mschad ∙�

CnHydroxyacylCoAMAT[t]

CnKetoacylCoAMAT[t] NADtMAT-NADHMAT[t] NADHMAT[t] + � ��∙�1+ 𝐾𝑚NADMAT 𝐾𝑚NADHMAT CnHydroxyacylCoAMAT[t] 𝐾𝑚CnKetoacylCoAMAT[t]

�1+∑Cn n→4,6,8,10,12,14 𝑎𝑛𝑑 16�𝐾𝑚

CnKetoacylCoAMAT[t]

CnAcylCoAMAT[t] AcetylCoAMAT[t] CoAMAT AcetylCoAMAT[t] + �+𝐾𝑚 �∙�1+𝐾𝑚 � CoAMAT 𝐾𝑚AcetylCoAMAT[t] CnKetoacylCoAMAT[t] 𝐾𝑚CnAcylCoAMAT[t] AcetylCoAMAT[t]

�1+∑Cn n→4,6,8,10,12,14 𝑎𝑛𝑑 16�𝐾𝑚

CnEnoylCoAMAT[t]

∙�1+

𝑣acesink = 𝐾𝑠acesink ∙ (AcetylCoAMAT[t] − 𝐾1acesink )

𝑣fadhsink = 𝐾𝑠fadhsink ∙ (FADHMAT[t] − 𝐾1fadhsink )

(53)

(54)

(55)

+

CnEnoylCoAMAT[t]∙�NADtMAT-NADHMAT[t]�∙CoAMAT Cn-2AcylCoAMAT[t]∙NADHMAT[t]∙AcetylCoAMAT[t] − � 𝐾𝑚CnEnoylCoAMAT[t] ∙𝐾𝑚NADMAT ∙𝐾𝑚CoAMAT 𝐾𝑚CnEnoylCoAMAT[t] ∙𝐾𝑚NADMAT ∙𝐾𝑚CoAMAT ∙𝐾𝑒𝑞mtp CnAcylCoAMAT[t] C6AcylCoAMAT[t] AcetoacetylCoAMAT[t] + �+𝐾𝑚 � CnEnoylCoAMAT[t] 𝐾𝑚CnAcylCoAMAT[t] C6AcylCoAMAT[t] 𝐾𝑖AcetoacetylCoAMAT

�1+∑Cn n→8,10,12,14 𝑎𝑛𝑑 16�𝐾𝑚

(52)

+

CnKetoacylCoAMAT[t]∙CoAMAT Cn-2AcylCoAMAT[t]∙AcetylCoAMAT[t] − � 𝐾𝑚CnKetoacylCoAMAT[t] ∙𝐾𝑚CoAMAT 𝐾𝑚CnKetoacylCoAMAT[t] ∙𝐾𝑚CoAMAT ∙𝐾𝑒𝑞mckat

𝑠𝑓mckatCn ∙𝑉mckat ∙�

𝑠𝑓mtpCn ∙𝑉mtp ∙�

(51)

+

CnAcylCoAMAT[t]∙�FADtMAT-FADHMAT[t]� CnEnoylCoAMAT[t]∙FADHMAT[t] − � 𝐾𝑚 ∙𝐾𝑚FADMAT 𝐾𝑚CnAcylCoAMAT[t] ∙𝐾𝑚FADMAT ∙𝐾𝑒𝑞scad C4-C6AcylCoAMAT[t]

𝑠𝑓scadCn ∙𝑉scad ∙�

(50)

+

(56)

+

NADtMAT-NADHMAT[t] NADHMAT[t] CoAMAT AcetylCoAMAT[t] + �∙�1+ + � 𝐾𝑚NADMAT 𝐾𝑚NADHMAT 𝐾𝑚CoAMAT 𝐾𝑚AcetylCoAMAT[t]

(57) (58)

𝑣nadhsink = 𝐾𝑠nadhsink ∙ (NADHMAT[t] − 𝐾1nadhsink )

(59)

CoAMAT = CoAMATt − ∑Cn n→8,10,12,14 𝑎𝑛𝑑 16(CnAcylCoAMAT[t] + CnEnoylCoAMAT[t] + CnHydroxyacylCoAMAT[t] + CnKetoacylCoAMAT[t]) − AcetylCoAMAT[t] (60)

3 Simulations for model validation The parameters in Table 1 below were used for steady-state calculations, unless parameter variations are indicated in the Figure. These steady-state calculations represent the functioning of mitochondria in the cell. To produce Figure 2 (the in vitro experiment with isolated mitochondria), the computer model was slightly adapted to match the conditions used in this experiment. In the experiment the supplied palmitoyl-CoA or palmitoyl-carnitine substrate decreased with time and this time course was imposed on the model, instead of the constant concentration above, which was used for steady state calculations. The substrate consumption dynamics was fitted to the concentration of palmitoyl carnitine over time, which resulted in the following equation: [𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒] = 26.8 ∙ 𝑒 −0.18∙𝑡

(61)

Here the substrate concentration is in µM and time t in minutes. As we could not measure

the time course for palmitoyl CoA, we used the same time course for palmitoyl CoA when it was given as a substrate. In the latter case palmitoyl carnitine was a free variable, predicted by the model and validated independently in the experiment. The concentrations of CarCYT was set to 400 µM, which was the average value measured over time. For VCYT we took 10-2 L.mgProtein-1, which here represents the extramitochondrial volume in the reaction vessel rather than the cytosolic volume. The remaining parameters were not changed. The initial concentrations of the acyl carnitines were set to the measured concentrations at time point 0.

Table 1: Kinetic parameters Parameter CPT1 sfcpt1C16 Vcpt1 Kmcpt1C16AcylCoACYT Kmcpt1CarCYT Kmcpt1C16AcylCarCYT Kmcpt1CoACYT Kicpt1MalCoACYT Keqcpt1 ncpt1

Value

Reference

1 0.012 13.8 250 136 40.7 9.1 0.45 2.4799

CACT Vfcact Vrcact KmC16AcylCarCYT KmCarMAT KmC16AcylCarMAT KmCarCYT KiC16AcylCarCYT KiCarCYT Keqcact

0.42 0.42 15 130 15 130 56 200 1

CPT2 sfcpt2C16 sfcpt2C14 sfcpt2C12 sfcpt2C10 sfcpt2C8 sfcpt2C6 sfcpt2C4 Vcpt2 KmCnAcylCarMAT KmCoAMAT KmCnAcylCoAMAT KmCarMAT Keqcpt2

0.85 1 0.95 0.95 0.35 0.15 0.01 0.391 51 30 38 350 2.22

VLCAD sfvlcadC16 sfvlcadC14 sfvlcadC12 Vvlcad KmC16AcylCoAMAT KmC14AcylCoAMAT KmC12AcylCoAMAT KmFAD KmC16EnoylCoAMAT KmC14EnoylCoAMAT KmC12EnoylCoAMAT KmFADH Keqvlcad

1 0.42 0.11 0.008 6.5 4 2.7 0.12 1.08 1.08 1.08 24.2 6

-1

µmol.min .mgProtein µM µM µM µM µM

-1

Fitted to experimental data

(2) (2) (2) (2) (3) (4) Estimated based on data of (5)

-1

-1

µmol.min .mgProtein -1 -1 µmol.min .mgProtein µM µM µM µM µM µM

-1

µmol.min .mgProtein µM µM µM µM

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM

-1

-1

(2) (2) (2) (2) (2) (2) Based on passive transport

(6) (6) (6) (6) (6) (6) (6) (6) (2) (2) (2) (2) (4) (7) (7) (7) Fitted to experimental data

(7) (7) (7) (1) (1) (1) (1) (1) (2)

Parameter

Value

Reference

LCAD sflcadC16 sflcadC14 sflcadC12 sflcadC10 sflcadC8 Vlcad KmC16AcylCoAMAT KmC14AcylCoAMAT KmC12AcylCoAMAT KmC10AcylCoAMAT KmC8AcylCoAMAT KmFAD KmC16EnoylCoAMAT KmC14EnoylCoAMAT KmC12EnoylCoAMAT KmC10EnoylCoAMAT KmC8EnoylCoAMAT KmFADH Keqlcad

0.9 1 0.9 0.75 0.4 0.01 2.5 7.4 9 24.3 123 0.12 1.08 1.08 1.08 1.08 1.08 24.2 6

MCAD sfmcadC12 sfmcadC10 sfmcadC8 sfmcadC6 sfmcadC4 Vmcad KmC12AcylCoAMAT KmC10AcylCoAMAT KmC8AcylCoAMAT KmC6AcylCoAMAT KmC4AcylCoAMAT KmFAD KmC12EnoylCoAMAT KmC10EnoylCoAMAT KmC8EnoylCoAMAT KmC6EnoylCoAMAT KmC4EnoylCoAMAT KmFADH Keqmcad

0.38 0.8 0.87 1 0.12 0.081 5.7 5.4 4 9.4 135 0.12 1.08 1.08 1.08 1.08 1.08 24.2 6

SCAD sfscadC6 sfscadC4 Vscad KmC6AcylCoAMAT KmC4AcylCoAMAT KmFAD KmC6EnoylCoAMAT KmC4EnoylCoAMAT KmFADH Keqscad

0.3 1 0.081 285 10.7 0.12 1.08 1.08 24.2 6

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM µM µM µM µM

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM µM µM µM µM

-1

µmol.min .mgProtein µM µM µM µM µM µM

-1

(8) (8) (8) (8) (8) Fitted to experimental data

(8) (8) (8) (8) (8) (1) (1) (1) (1) (1) (1) (1) (2)

-1

-1

(8) (8) (8) (8) (8) (8) (8) (8) (8) (8) (8) (1) (1) (1) (1) (1) (1) (1) (2) (8) (8) (8) (8) (8) (1) (1) (1) (1) (2)

Parameter

Value

Reference

CROT sfcrotC16 sfcrotC14 sfcrotC12 sfcrotC10 sfcrotC8 sfcrotC6 sfcrotC4 Vcrot KmC16EnoylCoAMAT KmC14EnoylCoAMAT KmC12EnoylCoAMAT KmC10EnoylCoAMAT KmC8EnoylCoAMAT KmC6EnoylCoAMAT KmC4EnoylCoAMAT KmC16HydroxyacylCoAMAT KmC14HydroxyacylCoAMAT KmC12HydroxyacylCoAMAT KmC10HydroxyacylCoAMAT KmC8HydroxyacylCoAMAT KmC6HydroxyacylCoAMAT KmC4HydroxyacylCoAMAT KiacetoacetylCoAMAT Keqcrot

0.13 0.2 0.25 0.33 0.58 0.8 1 3.6 150 100 25 25 25 25 40 45 45 45 45 45 45 45 1.6 3.13

M/SCHAD sfmschadC16 sfmschadC14 sfmschadC12 sfmschadC10 sfmschadC8 sfmschadC6 sfmschadC4 Vmschad KmC16HydroxyacylCoAMAT KmC14HydroxyacylCoAMAT KmC12HydroxyacylCoAMAT KmC10HydroxyacylCoAMAT KmC8HydroxyacylCoAMAT KmC6HydroxyacylCoAMAT KmC4HydroxyacylCoAMAT KmNADMAT KmC16KetoacylCoAMAT KmC14KetoacylCoAMAT KmC12KetoacylCoAMAT KmC10KetoacylCoAMAT KmC8KetoacylCoAMAT KmC6KetoacylCoAMAT KmC4AcetoacylCoAMAT KmNADHMAT Keqmschad

0.6 0.5 0.43 0.64 0.89 1 0.67 1 1.5 1.8 3.7 8.8 16.3 28.6 69.9 58.5 1.4 1.4 1.6 2.3 4.1 5.8 16.9 5.4 -4 2.17·10

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM

-1

(9) (9) (9) (9) (9) (9) (9) (9) (9) (9) (9) (9) (9) (9) (9) (2) (2) (2) (2) (2) (2) (2) (10) (2) (11) Estimated based on (11)

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM

-1

(11) (11) (11) (11) (11) (12) (13) (13) (13) (11) (11) (11) (11) (11) (13) (13) (13) (13) (13) (13) (11) (11) (2)

Parameter

Value

MCKAT sfmckatC16 sfmckatC14 sfmckatC12 sfmckatC10 sfmckatC8 sfmckatC6 sfmckatC4 Vmckat KmC16KetoacylCoAMAT KmC14KetoacylCoAMAT KmC12KetoacylCoAMAT KmC10KetoacylCoAMAT KmC8KetoacylCoAMAT KmC6KetoacylCoAMAT KmC4AcetoacylCoAMAT KmCoAMAT KmC16AcylCoAMAT KmC14AcylCoAMAT KmC12AcylCoAMAT KmC10AcylCoAMAT KmC8AcylCoAMAT KmC6AcylCoAMAT KmC4AcylCoAMAT KmAcetylCoAMAT Keqmckat

0 0.2 0.38 0.65 0.81 1 0.49 0.377 1.1 1.2 1.3 2.1 3.2 6.7 12.4 26.6 13.83 13.83 13.83 13.83 13.83 13.83 13.83 30 1051

MTP sfmtpC16 sfmtpC14 sfmtpC12 sfmtpC10 sfmtpC8 Vmtp KmC16EnoylCoAMAT KmC14EnoylCoAMAT KmC12EnoylCoAMAT KmC10EnoylCoAMAT KmC8EnoylCoAMAT KmNADMAT KmCoAMAT KmC16AcylCoAMAT KmC14AcylCoAMAT KmC12AcylCoAMAT KmC10AcylCoAMAT KmC8AcylCoAMAT KmC6AcylCoAMAT KmNADHMAT KmAcetylCoAMAT Keqmtp

1 0.9 0.81 0.73 0.34 2.84 25 25 25 25 25 60 30 13.83 13.83 13.83 13.83 13.83 13.83 50 30 0.71

Reference

(11) Estimated based on (11)

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM

-1

µmol.min .mgProtein µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM

-1

(11) (11) (11) (11) (11) (14) (15) Estimated based on (15)

(15) (15) (15) (15) (15) Estimated based on (15)

(2) (2) (2) (2) (2) (2) (2) (2) (2)

-1

(11) (11) (11) (11) (11) (11) (9) (9) (9) (9) (9) (11) (15) (2) (2) (2) (2) (2) (2) (11) (2)

Calculated by multiplying Keqcrot, Keqmschad and Keqmckat

Parameter

Value

Reference

ACESINK Ksacesink K1acesink

6000000 70

µmol.min .mgProtein µM

FADHSINK Ksfadhsink K1fadhsink

6000000 0.46


NADHSINK Ksnadhsink K1nadhsink

6000000 16


-1

-1

-1

-1

-1

-1

Total concentrations of the conserved moieties FADtMAT 0.77 µM NADtMAT 250 µM CoAMATt 5000 µM Fixed concentrations of metabolites CarCYT CoACYT CarMAT

200 140 950

Volumes of various compartments -6 VCYT 2.2x10 -6 VMAT 1.8x10

(1) (5) (1) (1) (1)

(1) (16)

µM µM µM

(1) (16) (1) -1

L.mgProtein -1 L.mgProtein

(17) (18)

Glossary venzymeCn SfenzymeCn

Venzyme KmCnmetabolite Keqenzyme (Cn)MetaboliteMAT[t]

(Cn)MetaboliteCYT[t] KiMetabolite ncpt1 FADtMAT NADtMAT CoAMATt Car CPT1 CACT CPT2 SCAD MCAD LCAD VLCAD CROT M/SCHAD MCKAT MTP VCYT VMAT vxsink Ksxsink K1xsink

Rate for a particular enzyme and carbon-chain length. For instance, vcpt1C16 is the rate at which the substrate with 16 C-atoms is converted by CPT1 Specificity factor that determines the enzyme activity for the substrate with a specific chain length as a percentage of the Vmax. The multiplication of this factor with Vmax will give the maximum enzyme rate for the substrate with n C-atoms. The Vmax of a particular enzyme. The affinity constant (Km) of an enzyme for the metabolite with a specific chain length, e.g. KmC16AcylCarCYT is the affinity constant of an enzyme for the acyl carnitine in the cytosol with 16 C-atoms. The equilibrium constant (Keq) for a particular enzyme reaction. Concentration of the metabolite in the mitochondrial matrix cytosol. When it starts with Cn, this denotes the chain length of the metabolite. The t in between brackets at the end depicts that the metabolite is a timedependent variable. Concentration of the metabolite in the cytosol. When it starts with Cn, this denotes the chain length of the metabolite. The t in between brackets at the end depicts that the metabolite is a time-dependent variable. Inhibition constant of an enzyme with respect to the metabolite. If the metabolite starts with Cn, this denotes the chain length of the metabolite. Hill coefficient of for the cooperative inhibition of CPT1 by malonyl-CoA. Total concentration of oxidized and reduced FAD in the mitochondrial matrix. Total concentration of oxidized and reduced NAD in the mitochondrial matrix. Total concentration of all CoA-containing species in the mitochondrial matrix. Carnitine. Carnitine-palmitoyl transferase 1. Carnitine-acyl-carnitine translocase. Carnitine-palmitoyl transferase 2. Short-chain acyl-CoA dehydrogenase. Medium-chain acyl-CoA dehydrogenase. Long-chain acyl-CoA dehydrogenase. Very-long-chain acyl-CoA dehydrogenase. Crotonase. Medium/short-chain hydroxyacyl-CoA dehydrogenase. Medium-chain ketoacyl-CoA thiolase. Mitochondrial trifunctional protein. Volume of the cytosol. Volume of the mitochondrial matrix. Rate of the sink reaction of metabolite x (x is either acetyl-CoA, NADH or FADH2). Rate constant of the sink reaction of metabolite x (x is either acetyl-CoA, NADH or FADH2). Constant in the sink reactions that determines the concentration of x (x is either acetyl-CoA, NADH or FADH2).

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16. Horie S, Isobe M & Suga T (1986) Changes in CoA pools in hepatic peroxisomes of the rat under various conditions. Journal of Biochemistry 99: 1345-1352. 17. Stoll B, Gerok W, Lang F & Häussinger D (1992) Liver cell volume and protein synthesis. The Biochemical Journal 287 ( Pt 1: 217-222. 18. Gear AR & Bednarek JM (1972) Direct counting and sizing of mitochondria in solution. The Journal of Cell Biology 54: 325-345.