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Accession Type:
Network
Ajay_Bhalla_
2004_Feedback_
Tuning
Shared_Object_
Ajay_Bhalla_
2004_Feedback_
Tuning
 Molecule
 Enzyme
 Reaction
PKC
PLA2
PLCbeta
Gq
MAPK
Ras
EGFR
Sos
PLC_g
CaMKII
CaM
PP1
PP2B
PKA
AC

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Enzyme List for pathway Shared_Object_Ajay_Bhalla_2004_Feedback_Tuning (Pathway Number 347)

 Molecule Name/
Site Name 		Km (uM) kcat (1/s)Ratio
(k2/k3)		Enzyme TypeSubstrate Product
1 Enzyme Activity:
craf**-deph

Enzyme Molecule:
PPhosphatase2A		15.656864explicit E-S complexcraf-1**
craf-1*
  Ueki et al JBC 269(22) pp 15756-15761 1994 show hyperphosphorylation of craf, so this is there to dephosphorylate it. Identity of phosphatase is not known to me, but it may be PP2A like the rest, so I have made it so.
2 Enzyme Activity:
craf-deph

Enzyme Molecule:
PPhosphatase2A		15.656864explicit E-S complexcraf-1*
craf-1
  See parent PPhosphatase2A for parms
3 Enzyme Activity:
Deph-thr286

Enzyme Molecule:
PP1-active		5.099110.354explicit E-S complex
  • CaMKII-thr286*-C
    aM
    		• CaMKII-CaM
      The rates are from Stralfors et al Eur J Biochem 149 295-303 giving Vmax = 5.7 umol/min giving k3 = 3.5/sec and k2 = 14. Foulkes et al Eur J Biochem 132 309-313 1983 give Km = 5.1 uM so k1 becomes 5.72e-6 Simonelli 1984 (Grad Thesis, CUNY) showed that other substrates are about 1/10 rate of phosphorylase a, so we reduce k1,k2,k3 by 10 to 5.72e-7, 1.4, 0.35
    4 Enzyme Activity:
    Deph-thr286c

    Enzyme Molecule:
    PP1-active    		5.099110.354explicit E-S complexCaMKII***
    		CaMK-thr306
     
    5 Enzyme Activity:
    Deph-thr305

    Enzyme Molecule:
    PP1-active    		5.099110.354explicit E-S complexCaMKII***
    		CaMKII-thr286
     
    6 Enzyme Activity:
    Deph-thr306

    Enzyme Molecule:
    PP1-active    		5.099110.354explicit E-S complexCaMK-thr306
    		CaMKII
      See Cohen et al
    7 Enzyme Activity:
    dephosph-PP1-I*

    Enzyme Molecule:
    CaM(Ca)n-CaNAB    		4.970760.344explicit E-S complexPP1-I1*
    		PP1-I1
     
    8 Enzyme Activity:
    dephosph_inhib1

    Enzyme Molecule:
    CaM(Ca)n-CaNAB    		4.970760.344explicit E-S complexI1*
    		I1
     
    9 Enzyme Activity:
    dephosph_
    inhib1_noCaM

    Enzyme Molecule:
    CaNAB-Ca4    		4.970760.0344explicit E-S complexI1*
    		I1
      The rates here are so slow I do not know if we should even bother with this enz reacn. These numbers are from Liu and Storm. Other refs suggest that the Km stays the same but the Vmax goes to 10% of the CaM stim levels. Prev: k1=2.2e-9, k2 = 0.0052, k3 = 0.0013 New : k1=5.7e-8, k2=.136, k3=.034
    10 Enzyme Activity:
    dephosph_
    neurogranin

    Enzyme Molecule:
    CaM(Ca)n-CaNAB    		10.01190.673.98507explicit E-S complexneurogranin*
    		neurogranin
      From Seki et al ABB 316(2):673-679
    11 Enzyme Activity:
    Deph_thr286b

    Enzyme Molecule:
    PP1-active    		5.099110.354explicit E-S complexCaMKII-thr286
    		CaMKII
     
    12 Enzyme Activity:
    MAPK*

    Enzyme Molecule:
    MAPK*    		25.6406104explicit E-S complexPLA2-cytosolic
    		PLA2*
      Km = 25uM @ 50 uM ATP and 1mg/ml MBP (huge XS of substrate) Vmax = 4124 pmol/min/ml at a conc of 125 pmol/ml of enz, so: k3 = .5/sec (rate limiting) k1 = (k2 + k3)/Km = (.5 + 0)/(25*6e5) = 2e-8 (#/cell)^-1 #s from Sanghera et al JBC 265 pp 52 , 1990. From Nemenoff et al JBC 268(3):1960-1964 - using Sanghera's 1e-4 ratio of MAPK to protein, we get k3 = 7/sec from 1000 pmol/min/mg fig 5
    13 Enzyme Activity:
    MAPK*-feedback

    Enzyme Molecule:
    MAPK*    		25.6406104explicit E-S complexcraf-1*
    		craf-1**
      Ueki et al JBC 269(22):15756-15761 show the presence of this step, but not the rate consts, which are derived from Sanghera et al JBC 265(1):52-57, 1990, see the deriv in the MAPK* notes.
    14 Enzyme Activity:
    MAPKK-deph

    Enzyme Molecule:
    PPhosphatase2A    		15.656864explicit E-S complexMAPKK*
    		MAPKK-ser
      See: Kyriakis et al Nature 358 pp 417-421 1992 Ahn et al Curr Op Cell Biol 4:992-999 1992 for this pathway. See parent PPhosphatase2A for parms.
    15 Enzyme Activity:
    MAPKK-deph-ser

    Enzyme Molecule:
    PPhosphatase2A    		15.656864explicit E-S complexMAPKK-ser
    		MAPKK
     
    16 Enzyme Activity:
    MKP1-thr-deph

    Enzyme Molecule:
    MKP-1    		0.13333144explicit E-S complexMAPK*
    		MAPK-tyr
      See MKP1-tyr-deph
    17 Enzyme Activity:
    MKP1-tyr-deph

    Enzyme Molecule:
    MKP-1    		0.13333144explicit E-S complexMAPK-tyr
    		MAPK
      The original kinetics have been modified to obey the k2 = 4 * k3 rule, while keeping kcat and Km fixed. As noted in the NOTES, the only constraining data point is the time course of MAPK dephosphorylation, which this model satisfies. It would be nice to have more accurate estimates of rate consts and MKP-1 levels from the literature. Effective Km : 67 nM kcat = 1.43 umol/min/mg
    18 Enzyme Activity:
    phosph-AC2

    Enzyme Molecule:
    PKC-active    		33.333244explicit E-S complexAC2
    		AC2*
      Phorbol esters have little effect on AC1 or on the Gs-stimulation of AC2. So in this model we are only dealing with the increase in basal activation of AC2 induced by PKC k1 = 1.66e-6 k2 = 16 k3 =4
    19 Enzyme Activity:
    phosph-PDE

    Enzyme Molecule:
    PKA-active    		7.4999694explicit E-S complexcAMP-PDE
    		cAMP-PDE*
      Same rates as PKA-phosph-I1
    20 Enzyme Activity:
    phosph_Sos

    Enzyme Molecule:
    MAPK*    		2.56406104explicit E-S complexSos
    		Sos*
      See Porfiri and McCormick JBC 271:10 pp5871 1996 for the existence of this step. We'll take the rates from the ones used for the phosph of Raf by MAPK. Sep 17 1997: The transient activation curve matches better with k1 up by 10 x.
    21 Enzyme Activity:
    PKA-phosph-GEF

    Enzyme Molecule:
    PKA-active    		7.4999694explicit E-S complexinact-GEF
    		inact-GEF*
      This pathway inhibits Ras when cAMP is elevated. See: Hordijk et al JBC 269:5 3534-3538 1994 Burgering et al EMBO J 12:11 4211-4220 1993 The rates are the same as used in PKA-phosph-I1
    22 Enzyme Activity:
    PKA-phosph-I1

    Enzyme Molecule:
    PKA-active    		7.4999694explicit E-S complexI1
    		I1*
      #s from Bramson et al CRC crit rev Biochem 15:2 93-124. They have a huge list of peptide substrates and I have chosen high-ish rates. These consts give too much PKA activity, so lower Vmax 1/3. Now, k1 = 3e-5, k2 = 36, k3 = 9 (still pretty fast). Also lower Km 1/3 so k1 = 1e-5 Cohen et al FEBS Lett 76:182-86 1977 say rate =30% PKA act on phosphokinase beta.
    23 Enzyme Activity:
    PKC-act-GEF

    Enzyme Molecule:
    PKC-active    		3.3333244explicit E-S complexinact-GEF
    		GEF*
      Rate consts from PKC-act-raf. This reaction activates GEF. It can lead to at least 2X stim of ras, and a 2X stim of MAPK over and above that obtained via direct phosph of c-raf. Note that it is a push-pull reaction, and there is also a contribution through the phosphorylation and inactivation of GAPs. The original PKC-act-raf rate consts are too fast. We lower K1 by 10 X
    24 Enzyme Activity:
    PKC-act-raf

    Enzyme Molecule:
    PKC-active    		66.667344explicit E-S complexcraf-1
    		craf-1*
      Rate consts from Chen et al Biochem 32, 1032 (1993) k3 = k2 = 4 k1 = 9e-5 recalculated gives 1.666e-5, which is not very different. Looks like k3 is rate-limiting in this case: there is a huge amount of craf locked up in the enz complex. Let us assume a 10x higher Km, ie, lower affinity. k1 drops by 10x. Also changed k2 to 4x k3. Lowerd k1 to 1e-6 to balance 10X DAG sensitivity of PKC
    25 Enzyme Activity:
    PKC-inact-GAP

    Enzyme Molecule:
    PKC-active    		3.3333244explicit E-S complexGAP
    		GAP*
      Rate consts copied from PCK-act-raf This reaction inactivates GAP. The idea is from the Boguski and McCormick review.
    26 Enzyme Activity:
    PKC-phosph-neuro
    granin

    Enzyme Molecule:
    PKC-active    		28.62830.584.03448explicit E-S complexneurogranin
    		neurogranin*
      Rates from Huang et al ABB 305:2 570-580 1993
    27 Enzyme Activity:
    PKC-phosph-ng-Ca
    M

    Enzyme Molecule:
    PKC-active    		28.59480.354explicit E-S complexneurogranin-CaM
    		CaM
    neurogranin*
      Rates are 60% those of PKC-phosph-neurogranin. See Huang et al ABB 305:2 570-580 1993
    28 Enzyme Activity:
    PP2A-dephosph-I1

    Enzyme Molecule:
    PP2A    		7.8282864.16667explicit E-S complexI1*
    		I1
      PP2A does most of the dephosph of I1 at basal Ca levels. See the review by Cohen in Ann Rev Biochem 1989. For now, lets halve Km. k1 was 3.3e-6, now 6.6e-6
    29 Enzyme Activity:
    PP2A-dephosph-PP
    1-I*

    Enzyme Molecule:
    PP2A    		7.8282864.16667explicit E-S complexPP1-I1*
    		PP1-I1
      k1 changed from 3.3e-6 to 6.6e-6
    30 Enzyme Activity:
    Sos.Ras_GEF

    Enzyme Molecule:
    Shc*.Sos.Grb2    		0.5050510.024explicit E-S complexGDP-Ras
    		GTP-Ras
     


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