(*Mitochondrial Deoxyribonucleoside Salvage Pathway*)
(*Vishal V Gandhi and David C Samuels*)

(*DeoxynucleotideModelConstants.txt*)
(*This file is the constants file for the Mathematica*)
(*mitochondrial deoxynucleotide metabolism and mtDNA*)
(*synthesis model*)

Lstrandstart=10969;

(*the fractions of A,C,T, and G on the heavy and light strands of mtDNA*)
fdTH=0.309;
fdTL=0.247;
fdCH=0.131;
fdCL=0.313;
fdAH=0.247;
fdAL=0.309;
fdGH=0.313;
fdGL=0.131;

(*the Hill coefficient of TK2 for thymidine*)
tk2hill=0.5;

(*The length of both strands of mtDNA*)
DNAlength=33136;
(*the length of one strand of mtDNA*)
strandDNA=DNAlength/2;

(*volume of a mitochondrion*)
volmito=2*^-16;

(*conversion factor used to convert kms and concentrations from microMolar to molecules/mitochondrion*)
(*conversion = 120.4;*)
conversion=1*^-6*6.022*^23*volmito;
secondsperminute=60;

(*factor used to decrease the vmax of the polymerase on double stranded templates with lower primer density*)
dsfact=1/2;

(*Polymerase kinetic constants Johnson 01 JBC*)
VmaxPoldT=25.0*dsfact*secondsperminute;
VmaxPoldC=43.0*dsfact*secondsperminute;
VmaxPoldA=45.0*dsfact*secondsperminute;
VmaxPoldG=37.0*dsfact*secondsperminute;
KmPoldT=0.63*conversion;
KmPoldC=0.9*conversion;
KmPoldA=0.8*conversion;
KmPoldG=0.8*conversion;

(*Ki of dTTP on tk2 Wang 03 DOI 10.1074/jbc.M206143200*)
kidttptk2=2.3*conversion;

(*Ki of dUTP on tk2 Geometric mean of dCTP and dTTP values*)
kidutptk2=1.38*conversion;

(*Ki of dCTP on tk2 Wang 03 DOI 10.1074/jbc.M206143200*)
kidctptk2=0.83*conversion;

(*Ki of dU on tk2 Geometric mean Munch-Petersen 91 JBC*)
kidutk2=227*conversion;

(*Ki of dC on tk2 Wang 03 DOI 10.1074/jbc.M206143200*)
kidctk2=40*conversion;

(*Ki of dT on tk2 Wang 03 DOI 10.1074/jbc.M206143200*)
kidttk2=4.9*conversion;

(*Substrate Kis on dgk set equal to substrate kms*)

(*Ki of dI on dgk set equal to km Sjoberg 98 Molecular Pharmacology*)
kididgk=12*conversion;

(*Sjoberg 01 DOI: 10.1128/AAC.45.3.739–742.2001, Ki of dITP on dgk set equal to dATP Ki*)
kidimpdgk=78*conversion;
kiditpdgk=kidatpdgk;

(*Ki of dGMP on dgk Sjoberg 01 DOI: 10.1128/AAC.45.3.739–742.2001*)
kidgmpdgk=4*conversion;

(*Ki of dAMP on dgk Sjoberg 01 DOI: 10.1128/AAC.45.3.739–742.2001*)
kidampdgk=28*conversion;

(*Ki of dATP on dgk Sjoberg 01 DOI: 10.1128/AAC.45.3.739–742.2001*)
kidatpdgk=41*conversion;

(*Ki of dGTP on dgk Sjoberg 01 DOI: 10.1128/AAC.45.3.739–742.2001*)
kidgtpdgk=0.4*conversion;

(*estimated nucleoside transporter molecular weight in kD Griffiths 97 Nature Medicine assumed monomer?*)
transporterMW=50;

(*tk2 and dgk molecular weight in kD=29 Wang 99 Febs Letters, Mandel 01 doi:10.1038/ng746*)
(*dgk is a dimer, tk2 exists both as dimer and tetramer: tetramer is more active but less abundant, transition between the 2 states is possible and ATP-mediated (mean taken)*)
dgkMW=58;
tk2MW=87;

(*molecular weight of dnt2 in kD Rampazzo 00 PNAS Hunsucker 05 dimer = 2*23*)
dnt2MW=46;

(*Ectonucleotidase molecular weight Tetramer Brenda*)
enMW=210;

(*tmpk2 molecular weight in kD Chen 08 Genes to Cells*)
tmpk2MW=44;

(*gmpk2 molecular weight in kD Brenda*)
gmpk2MW=22;

(*cmpk2 molecular weight in kD Xu08 JBC*)
cmpk2MW=44.5;

(*ak2 molecular weight in kD Uniprot/other literature*)
akMW=26;

(*human nme4 molecular weight in kD=20 Uniprot/Milon 00, homohexamer*)
ndpkMW=120;

(*nucleoside kinase molecules in each mitochondrion from Saada 01 and 03 Nature Genetics and Mol Gen Metabolism*)
(*as much as 20-fold variation may exist between tissues*)
tk2moleculespermito=100;
dgkmoleculespermito=200;

(*dnt2 molecules in each mitochondrion*)
dnt2moleculespermito=50;

(*Ectonucleotidase molecules in each mitochondrion*)
enmoleculespermito=50;

(*tmpk2 molecules in each mitochondrion*)
tmpk2moleculespermito=50;

(*gmpk2 molecules in each mitochondrion*)
gmpk2moleculespermito=50;

(*cmpk2 molecules in each mitochondrion*)
cmpk2moleculespermito=50;

(*ndpk molecules in each mitochondrion*)
ndpkmoleculespermito=300;

(*the factor that the reverse reaction is faster than the forward reaction for NMPK*)
factorMD=0.1;(*AMP/ADP*)

(*the factor that the reverse reaction is faster than the forward reaction for NDPK*)
factorDT=0.1;(*ADP/ATP*)

(*ent molecules per mitochondrion Life Sciences Camins 96, Escubedo 00*)
transportermoleculespermito=38;

(*adenylate kinase molecules per mitochondrion Eur. J. Biochem. 93, 263 1979 Tomaselli*)
akmoleculespermito=450;

(*number of total proteins in a mitochondrion assuming average MW of 30 kD and 5x10^10 mito/mg mito protein*)
(*proteinspermito=400000;*)

(*transporter Vmax converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute refer base model constants, camins 95, jimenez 00*)
transportervmax=0.000086/0.0000021*transportermoleculespermito;

(*agreement between kcat from gerth 07 for tk2 at least and the Vmax values - so ok*)

(*Vmax of the first phosphorylation of dT in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Wang 03 DOI 10.1074/jbc.M206143200*)
Vmax1PfdT=1.288*tk2MW*tk2moleculespermito;

(*Vmax of the first phosphorylation of dC in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Wang 03 DOI 10.1074/jbc.M206143200*)
Vmax1PfdC=0.789*tk2MW*tk2moleculespermito;

(*Vmax of dC with dgk converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Sjoberg 98 Molecular Pharmacology*)
Vmax1PfdCdgk=0.059*dgkMW*dgkmoleculespermito;

(*Vmax of the first phosphorylation of dA in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Sjoberg 98 Molecular Pharmacology*)
Vmax1PfdA=0.429*dgkMW*dgkmoleculespermito;

(*Vmax of the first phosphorylation of dG in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Sjoberg 98 Molecular Pharmacology*)
Vmax1PfdG=0.043*dgkMW*dgkmoleculespermito;

(*turnover numbers for cytosolic nucleotidases from Brenda seem to match Vmax below*)

(*Vmax of the first phosphorylation of dT in the reverse direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Mazzon 03 Biochemical Pharmacology*)
Vmax1PrdT=74*dnt2MW*dnt2moleculespermito;

(*Various sources, Spychala 89 for Vmax 45 for AMP so setting lower here, also refer Hunsucker 05*)

(*Ectonucleotidase Vmax of the first phosphorylation of dT in the reverse direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute*)
Vmax1PrdTen=4.5*enMW*enmoleculespermito;

(*Vmax of the first phosphorylation of dC in the reverse direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute*)
Vmax1PrdC=4.5*enMW*enmoleculespermito;

(*Vmax of the first phosphorylation of dA in the reverse direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute*)
Vmax1PrdA=4.5*enMW*enmoleculespermito;

(*Vmax of the first phosphorylation of dG in the reverse direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute*)
Vmax1PrdG=4.5*enMW*enmoleculespermito;

(*Vmax of the second phosphorylation of dT in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Brenda, also refer Pasti 03 Kcat value for cytoplasmic enzyme is 1 per second*)
Vmax2PfdT=0.821*tmpk2MW*tmpk2moleculespermito;

(*Vmax of the second phosphorylation of dC in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Xu 08 JBC*)
Vmax2PfdC=1.77*cmpk2MW*cmpk2moleculespermito;

(*Vmax of the second phosphorylation of dA in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Alexandre 07 Nucleic Acids Research*)
Vmax2PfdA=272.8*akMW*akmoleculespermito;

(*Vmax of the second phosphorylation of dG in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute from Brenda mouse and rat unreliable values* Hall 86 Eur J Biochem unreliable value*)
Vmax2PfdG=1.54*gmpk2MW*gmpk2moleculespermito;

(*Vmax of the second phosphorylation of dT in the reverse direction*)
Vmax2PrdT=Vmax2PfdT*factorMD;

(*Vmax of the second phosphorylation of dC in the reverse direction*)
Vmax2PrdC=Vmax2PfdC*factorMD;

(*Vmax of the second phosphorylation of dA in the reverse direction*)
Vmax2PrdA=Vmax2PfdA*factorMD;

(*Vmax of the second phosphorylation of dG in the reverse direction*)
Vmax2PrdG=Vmax2PfdG*factorMD;

(*Milon 00 human and Lambeth 97 pigeon both have data but Lambeth 97 has more, and there is overlap between dTDP values - so using Lambeth 97 for all nme4 data*)

(*Vmax of the third phosphorylation of dT in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Lambeth 97 JBC*)
Vmax3PfdT=140*ndpkMW*ndpkmoleculespermito;

(*Vmax of the third phosphorylation of dC in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Lambeth 97 JBC*)
Vmax3PfdC=50*ndpkMW*ndpkmoleculespermito;(*author statement: dNDPs are slower than rNDPs, so taking dCDP Vmax=CDP*)

(*Vmax of the third phosphorylation of dA in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Milon 00 JBC*)
Vmax3PfdA=225*ndpkMW*ndpkmoleculespermito;(*set equal to dGDP Vmax*)

(*Vmax of the third phosphorylation of dG in the forward direction converting from micromoles substrate/mg enzyme/minute to molecules substrate/mitochondrion/minute Lambeth 97 JBC*)
Vmax3PfdG=225*ndpkMW*ndpkmoleculespermito;

(*Vmax of the third phosphorylation of dT in the reverse direction*)
Vmax3PrdT=Vmax3PfdT*factorDT;

(*Vmax of the third phosphorylation of dC in the reverse direction*)
Vmax3PrdC=Vmax3PfdC*factorDT;

(*Vmax of the third phosphorylation of dA in the reverse direction*)
Vmax3PrdA=Vmax3PfdA*factorDT;

(*Vmax of the third phosphorylation of dG in the reverse direction*)
Vmax3PrdG=Vmax3PfdG*factorDT;

(*transporter Km Escubedo 00*)
transporterkm=2*conversion;

(*Km of the first phosphorylation of dT in the forward direction Wang 03 DOI 10.1074/jbc.M206143200*)
km1PfdT=13*conversion;

(*Km of the first phosphorylation of dC in the forward direction Wang 03 DOI 10.1074/jbc.M206143200*)
km1PfdC=11*conversion;

(*Km of dC with dgk Sjoberg 98 Molecular Pharmacology*)
km1PfdCdgk=336*conversion;

(*Km of the first phosphorylation of dA in the forward direction Sjoberg 98 Molecular Pharmacology*)
km1PfdA=467*conversion;

(*Km of the first phosphorylation of dG in the forward direction Sjoberg 98 Molecular Pharmacology*)
km1PfdG=4*conversion;

(*Km of the first phosphorylation of dT, dU in the reverse direction Rampazzo 00 PNAS*)
km1PrdT=200*conversion;
km1PrdU=100*conversion;
km1PrrU=1.5*km1PrdT;

(*Ectonucleotidase data from Hunsucker 05 or Brenda*)
(*Geometric means for substrate Kms, higher Kms plugged for inhibitions to be conservative*)

(*Ectonucleotidase Km of the first phosphorylation of dT, dU, rU in the reverse direction*)
km1PrdTen=22.5*conversion;
km1PrdUen=110*conversion;(*set equal to UMP Km*)
km1PrrUen=110*conversion;(*set equal to Km*)

(*Ectonucleotidase Km of the first phosphorylation of dC, rC in the reverse direction*)
km1PrdC=290*conversion;
km1PrrC=360*conversion;

(*Ectonucleotidase Km of the first phosphorylation of da, rA in the reverse direction*)
km1PrdA=62*conversion;
km1PrrA=19*conversion;(*set equal to Km*)

kiadpen=17*conversion;
kiatpen=15*conversion;

(*Ectonucleotidase Km of the first phosphorylation of dG, rG in the reverse direction*)
km1PrdG=48*conversion;
km1PrrG=59*conversion;(*set equal to Km*)

(*Ectonucleotidase Km of the first phosphorylation of dI, rI in the reverse direction*)
km1PrdI=100*conversion;(*set equal to Km of IMP*)
km1PrrI=100*conversion;(*set equal to Km*)

(*Km of the second phosphorylation of dT in the forward direction Alexandre 07,misc*)
km2PfdT=20*conversion;
km2PfdUtmpk2=2600*conversion;(*Km is 170, but Ki is 2600*)

(*miscellaneous inhibitions Brenda*)
(*thymidine inhibition excluded because even at 770 uM only 27% inhibition observed*)
kidttptmpk2=700*conversion;
kidttmpk2=180*conversion;

(*Km of the second phosphorylation of dC in the forward direction Xu 08*)
km2PfdC=1310*conversion;
km2PfrC=3090*conversion;
km2PfrU=6300*conversion;
km2PfdUcmpk2=100*conversion;

(*Refer VanRompay 99 Molecular Pharmacology cmpk1 can phosphorylate AMP and dAMP*)
km2PfrAcmpk2=km2PrrAcmpk2=km2PfdAcmpk2=km2PrdAcmpk2=100*500*conversion; (*km of CMP is 500 uM*)

(*Km of the second phosphorylation of dA in the forward direction Alexandre 07 Nucleic Acids Research*)
km2PfdA=210*conversion;
km2PfrA=80*conversion;(*Km is 80, Ki is 500 - but this gives the incorrect impression that dAMP is a better substrate*)

(*Refer Alexandre 07 Nucleic Acids Research 07 - CMP and UMP have some reactivity with ak2 - included as inhibitions*)
km2PfrCak2=6000*conversion;
km2PfrUak2=9000*conversion;

(*Km of the second phosphorylation of dG in the forward direction, Brenda*)
km2PfdG=112*conversion;
km2PfrG=18*conversion;

(*Km of the second phosphorylation of dT in the reverse direction*)
km2PrdT=km2PfdT;
km2PrdUtmpk2=km2PfdUtmpk2;

(*Km of the second phosphorylation of dC in the reverse direction*)
km2PrdC=km2PfdC;
km2PrrC=km2PfrC;
km2PrrU=km2PfrU;
km2PrdUcmpk2=km2PfdUcmpk2;

(*Km of the second phosphorylation of dA in the reverse direction*)
km2PrdA=km2PfdA;
km2PrrA=km2PfrA;

km2PrrCak2=km2PfrCak2;
km2PrrUak2=km2PfrUak2;

(*Km of the second phosphorylation of dG in the reverse direction*)
km2PrdG=km2PfdG;
km2PrrG=km2PfrG;

(*Reaction is linear for dTDP and UDP until at least 1000 uM Lambeth 97 JBC*)
(*Km of the third phosphorylation of dT in the forward direction Lambeth 97 JBC*)
km3PfdT=1000*conversion;
km3PfdU=km3PfdT;
km3PfrU=km3PfdT;

(*Km of the third phosphorylation of dC in the forward direction Lambeth 97 JBC*)
km3PfdC=1000*conversion;(*dNDPs are weaker substrates than rNDPs: author statement but data n/a so same value used*)
km3PfrC=1000*conversion;(*Reaction linear until at least 1000 uM*)

(*Km of the third phosphorylation of dA in the forward direction Lambeth 97 JBC*)
km3PfdA=70*conversion;(*Km of ADP is about 70 uM OR Km of dADP set equal to that of dGDP*)
km3PfrA=300*conversion;(*substrate inhibition, Ki*)

(*Km of the third phosphorylation of dG in the forward direction Lambeth 97 JBC*)
km3PfdG=75*conversion;
km3PfrG=100*conversion;(*substrate inhibition,Ki*)

(*inosine inhibitions*)
km3PfrI=km3PrrI=km3PfdI=km3PrdI=1000*conversion;

(*Km of the third phosphorylation of dT in the reverse direction*)
km3PrdT=km3PfdT;
km3PrdU=km3PrdT;
km3PrrU=km3PrdT;

(*Km of the third phosphorylation of dC in the reverse direction*)
km3PrdC=km3PfdC;
km3PrrC=km3PrdC;

(*Km of the third phosphorylation of dA in the reverse direction*)
km3PrdA=km3PfdA;
km3PrrA=km3PrdA;

(*Km of the third phosphorylation of dG in the reverse direction*)
km3PrdG=km3PfdG;
km3PrrG=km3PrdG;

(*initial concentrations*)

dTcyto=RandomReal[{0.05*conversion, 5*conversion}];
dCcyto=RandomReal[{0.05*conversion, 5*conversion}];
dAcyto=RandomReal[{0.05*conversion, 5*conversion}];
dGcyto=RandomReal[{0.05*conversion, 5*conversion}];

dT0=dTcyto;
dC0=dCcyto;
dA0=dAcyto;
dG0=dGcyto;

(*initial dNTP levels*)

(*for transport model, have set these to be chosen randomly*)

If[celltype==1,dTTPcyto=RandomReal[{0.1*conversion, 10*conversion}]];
If[celltype==1,dCTPcyto=RandomReal[{0.1*conversion, 10*conversion}]];
If[celltype==1,dATPcyto=RandomReal[{0.1*conversion, 10*conversion}]];
If[celltype==1,dGTPcyto=RandomReal[{0.1*conversion, 10*conversion}]];

dTMP0=RandomReal[{0.1*conversion, 10*conversion}];
dTDP0=RandomReal[{0.1*conversion, 10*conversion}];
dTTP0=dTTPcyto;

dCMP0=RandomReal[{0.1*conversion, 10*conversion}];
dCDP0=RandomReal[{0.1*conversion, 10*conversion}];
dCTP0=dCTPcyto;

dAMP0=RandomReal[{0.1*conversion, 10*conversion}];
dADP0=RandomReal[{0.1*conversion, 10*conversion}];
dATP0=dATPcyto;

dGMP0=RandomReal[{0.1*conversion, 10*conversion}];
dGDP0=RandomReal[{0.1*conversion, 10*conversion}];
dGTP0=dGTPcyto;

dU=dUcyto=dTcyto;
rU=rUcyto=dTcyto;
dI=dIcyto=0.1*dAcyto;
rI=rIcyto=0.1*dAcyto;
rC=rCcyto=dCcyto;
rA=rAcyto=dAcyto;
rG=rGcyto=dGcyto;

dUMP=0.1*dTMP0;
rUMP=10*dTMP0;
dIMP=0.1*dAMP0;
rIMP=0.1*dAMP0;
rCMP=10*dCMP0;
rAMP=10*dAMP0;
rGMP=10*dGMP0;

dUDP=0.1*dTDP0;
rUDP=10*dTDP0;
dIDP=0.1*dADP0;
rIDP=0.1*dADP0;
rCDP=10*dCDP0;
rADP=10*dADP0;
rGDP=10*dGDP0;

dUTP=0.1*dTTP0;
rUTP=10*dTTP0;
dITP=0.1*dATP0;
rITP=0.1*dATP0;
rCTP=10*dCTP0;
rATP=10*dATP0;
rGTP=10*dGTP0;

DNA0=0;
LDNA0=0;
HDNA0=0;

(*end file*)