| |
Reaction
Name | Pathway Name /
Pathway No. | Kf | Kb | Kd | tau | Reagents |
| 1 | Shc_bind_
Sos.Grb2 | Sos
Pathway No. 78 | 0.5
(uM^-1 s^-1) | 0.1
(s^-1) | Kd(bf) = 0.2(uM) | - | Substrate:
Sos.Grb2
SHC*
Products:
Shc*.Sos.Grb2
|
| Sasaoka et al JBC 269:51 pp 32621 1994, table on pg 32623 indicates that this pathway accounts for about 50% of the GEF activation. (88% - 39%). Error is large, about 20%. Fig 1 is most useful in constraining rates. Chook et al JBC 271:48 pp 30472, 1996 say that the Kd is 0.2 uM for Shc binding to EGFR. The Kd for Grb direct binding is 0.7, so we'll ignore it. | | 2 | Grb2_bind_Sos* | Sos
Pathway No. 78 | 0.025
(uM^-1 s^-1) | 0.0168
(s^-1) | Kd(bf) = 0.672(uM) | - | Substrate:
Sos*
Grb2
Products:
Sos*.Grb2
|
| Same rates as Grb2_bind_Sos: Porfiri and McCormick JBC 271:10 pp 5871 1996 show that the binding is not affected by the phosphorylation. | | 3 | dephosph_Sos | Sos
Pathway No. 78 | 0.001
(s^-1) | 0
(s^-1) | - | - | Substrate:
Sos*
Products:
Sos
|
| The best clue I have to these rates is from the time courses of the EGF activation, which is around 1 to 5 min. The dephosph would be expected to be of the same order, perhaps a bit longer. Lets use 0.002 which is about 8 min. Sep 17: The transient activation curve matches better with kf = 0.001 | | 4 | Grb2_bind_Sos | Sos
Pathway No. 78 | 0.025
(uM^-1 s^-1) | 0.0168
(s^-1) | Kd(bf) = 0.672(uM) | - | Substrate:
Grb2
Sos
Products:
Sos.Grb2
|
| As there are 2 SH3 domains, this reaction could be 2nd order. I have a Kd of 22 uM from peptide binding (Lemmon et al JBC 269:50 pg 31653). However, Chook et al JBC 271:48 pg30472 say it is 0.4uM with purified proteins, so we believe them. They say it is 1:1 binding. Porfiri and McCormick JBC 271 also have related data. After comparing with the time-course of 1 min and the efficacy of activation of Ras, settle on Kd of 0.672 which is close to the Chook et al value. | | 5 | Ras-act-craf | Shared_Object_
Synaptic_
Network
Pathway No. 70 | 24
(uM^-1 s^-1) | 0.5
(s^-1) | Kd(bf) = 0.0208(uM) | - | Substrate:
craf-1*
GTP-Ras
Products:
Raf-GTP-Ras*
|
| Assume binding is fast and limited only by available Ras*. So kf = kb/[craf-1] If kb is 1/sec, then kf = 1/0.2 uM = 1/(0.2 * 6e5) = 8.3e-6 Later: Raise it by 10 X to about 1e-4, giving a Kf of 60 for Kb of 0.5 and a tau of approx 2 sec. Based on: Hallberg et al JBC 269:6 3913-3916 1994, 3% of cellular Raf is complexed with Ras. This step needed to memb-anchor and activate Raf: Leevers et al Nature 369 411-414. Also see Koide et al 1993 PNAS USA 90(18):8683-8686 | | 6 | remove_glu | Shared_Object_
Synaptic_
Network
Pathway No. 70 | 500
(s^-1) | 1000
(s^-1) | Keq = 2(uM) | 0.001sec | Substrate:
Glu
Products:
synapse
|
| This reaction doubles for arrival as well as removal of glu from the synapse. Assume tau for removal of glu is ~1 msec. We know that diffusion time for arrival of glu from presynaptic side is < 50 usec. Most of the actual synaptic delay has to do with binding to the receptors. | | 7 | bg-act-GEF | Ras
Pathway No. 76 | 6
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 0.1667(uM) | - | Substrate:
BetaGamma
inact-GEF
Products:
GEF-Gprot-bg
|
| SoS/GEF is present at 50 nM ie 3e4/cell. BetaGamma maxes out at 9e4. Assume we have 1/3 of the GEF active when the BetaGamma is 1.5e4. so 1e4 * kb = 2e4 * 1.5e4 * kf, so kf/kb = 3e-5. The rate of this equil should be reasonably fast, say 1/sec | | 8 | dephosph-GEF | Ras
Pathway No. 76 | 1
(s^-1) | 0
(s^-1) | - | - | Substrate:
GEF*
Products:
inact-GEF
|
| This rate is based on the known ratio of GDP-Ras to GTP-Ras. Basal: Ras.GTP = 7% Stimulated 15% Time course is within 10 min, probably much faster as not all early data points are there. See Gibbs et al JBC 265(33):20437-20422 | | 9 | Ras-intrinsic-GT
Pase | Ras
Pathway No. 76 | 0.0001
(s^-1) | 0
(s^-1) | - | - | Substrate:
GTP-Ras
Products:
GDP-Ras
|
| This is extremely slow (kf = 1e-4), but it is significant as so little GAP actually gets complexed with it that the total GTP turnover rises only by 2-3 X (see Gibbs et al, JBC 265(33) 20437-20422) and Eccleston et al JBC 268(36) 27012-27019 There is no back reaction as we assume this to be a regular irreversible Michaelis-Menten zeroth order hydrolysis. | | 10 | dephosph-GAP | Ras
Pathway No. 76 | 0.1
(s^-1) | 0
(s^-1) | - | - | Substrate:
GAP*
Products:
GAP
|
| Assume a reasonably good rate for dephosphorylating it, 0.1/sec. This fits well with resting levels of active kinase and the degree of activation as well as time-course of turnoff of Ras activation, but data is quite indirect. | | 11 | CaM-bind-GEF | Ras
Pathway No. 76 | 60
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 0.0167(uM) | - | Substrate:
inact-GEF
CaM-Ca4
Products:
CaM-GEF
|
| We have no numbers for this. It is probably between the two extremes represented by the CaMKII phosph states, and I have used guesses based on this. kf=1e-4 kb=1 The reaction is based on Farnsworth et al Nature 376 524-527 1995 | | 12 | dephosph-inact-G
EF* | Ras
Pathway No. 76 | 1
(s^-1) | 0
(s^-1) | - | - | Substrate:
inact-GEF*
Products:
inact-GEF
|
| 13 | Ca-bind-CaNAB-Ca
2 | PP2B
Pathway No. 83 | 3.6
(uM^-2 s^-1) | 1
(s^-1) | Kd(af) = 0.527(uM) | - | Substrate:
Ca
Ca
CaNAB-Ca2
Products:
CaNAB-Ca4
|
| This process is probably much more complicated and involves CaM. However, as I can't find detailed info I am bundling this into a single step. Based on Steemer and Klee 1994 Biochem 33:6859-6866, this specific parm on pg 6863, the Kact is 0.5 uM. Assume binding is fast, 1 sec. | | 14 | Ca-bind-CaNAB | PP2B
Pathway No. 83 | 10008
(uM^-2 s^-1) | 1
(s^-1) | Kd(af) = 0.01(uM) | - | Substrate:
CaNAB
Ca
Ca
Products:
CaNAB-Ca2
|
| going on the experience with CaM, we put the fast (high affinity) sites first. We only know (Stemmer and Klee) that the affinity is < 70 nM. Assuming 10 nM at first. This doesn't really matter much because it will always be bound at physiological Ca. | | 15 | CaM-Ca2-bind-CaN
AB | PP2B
Pathway No. 83 | 0.24
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 4.1667(uM) | - | Substrate:
CaNAB-Ca4
CaM-TR2-Ca2
Products:
CaMCa2-CANAB
|
| Based on Stemmer and Klee 1994 Biochem 33:6859-6866 This model is actually a simplification of the detail they report. In this model the Ca-binding affinity of CaM is not changed by binding to CaN. Rates here come from a detailed-balance argument. The reference rate is for CaMCa4 binding to CaNAB. This rate should be 1/2500 of that. | | 16 | CaMCa3-bind-CaNA
B | PP2B
Pathway No. 83 | 2.238
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 0.4468(uM) | - | Substrate:
CaM-Ca3
CaNAB-Ca4
Products:
CaMCa3-CaNAB
|
| Rates derived from the CaMCa4 binding to CaNAB-Ca4 step. Due to detailed balance calculations taking the Ca affinity for CaM into account, this reaction should be 250 times slower. Close. | | 17 | CaMCa4-bind-CaNA
B | PP2B
Pathway No. 83 | 600
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 0.0017(uM) | - | Substrate:
CaM-Ca4
CaNAB-Ca4
Products:
CaMCa4-CaNAB
|
| This step is the starting point for calculating all the CaM-binding steps to CaNAB-Ca4. The calculation goes like this: From Stemmer and Klee 1994 Biochem 33 6859-6866 we have rates for Ca binding to CaM.Ca4.CaN. From detailed balance (Kd must be 1 around a loop) we can set ratios of Kds for CaMCa3 binding to CaN, and CaM-Ca2 binding to CaN. Thus those rates can come once we know the Kd for the current reaction of CaMCa4 binding to CaN. We'll ignore the Ca binding steps to CaM.Ca4.CaN since the reactions around the remaining part of the loop will settle pretty fast to the same levels. Finally, we estimate the kf=0.001 here from a series of simulations matching the curves in Stemmer and Klee. | | 18 | Inact-PP1 | PP1
Pathway No. 82 | 499.98
(uM^-1 s^-1) | 0.1
(s^-1) | Kd(bf) = 0.0002(uM) | - | Substrate:
I1*
PP1-active
Products:
PP1-I1*
|
| K inhib = 1nM from Cohen Ann Rev Bioch 1989, 4 nM from Foukes et al Assume 2 nM. kf /kb = 8.333e-4 The Kd used here is 0.2 nM. This is small, but unlikely to matter much as the affinity is so strong that the reaction will be all the way forward in either case. Tau < 1 min for inhibition. Stralfors 1985 Eur J Biochem 149:295-303 fig 8 pg 201. | | 19 | dissoc-PP1-I1 | PP1
Pathway No. 82 | 1
(s^-1) | 0
(uM^-1 s^-1) | - | - | Substrate:
PP1-I1
Products:
PP1-active
I1
|
| Assumption is that the affinity of the unphosphorylated form of I1 for PP1 is extremely weak and that the reaction is essentially all the way forward. The tau is fast at 1 sec. | | 20 | Ca_act_PLC_g | PLC_g
Pathway No. 79 | 180
(uM^-1 s^-1) | 10
(s^-1) | Kd(bf) = 0.0556(uM) | - | Substrate:
PLC_g
Ca
Products:
Ca.PLC_g
|
| Nice curves from Homma et al JBC 263:14 6592-6598 1988 Fig 5c. The activity falls above 10 uM, but that is too high to reach physiologically anyway, so we'll ignore the higher pts and match the lower ones only. Half-max at 1 uM. But Wahl et al JBC 267:15 10447-10456 1992 have half-max at 56 nM which is what I'll use. |
and 
color.
