| | Name | Accession Name | Pathway Name | Kf | Kb | Kd | tau | Reagents |
| 1 | PKC-act-by-Ca | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PKC
Pathway No. : 313 | 0.6
(uM^-1 s^-1) | 0.5
(s^-1) | Kd(bf) = 0.8333(uM) | - | Substrate
Ca
PKC-cytosolic
Product
PKC-Ca
|
| | Need est of rate of assoc of Ca and PKC. Assume it is fast The original parameter-searched kf of 439.4 has been scaled by 1/6e8 to account for change of units to n. Kf now 8.16e-7, kb=.6085 Raised kf to 1e-6 to match Ca curve, kb to .5 |
| 2 | PLA2-Ca-act | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PLA2
Pathway No. : 314 | 1
(uM^-1 s^-1) | 0.1
(s^-1) | Kd(bf) = 0.1(uM) | - | Substrate
Ca
PLA2-cytosolic
Product
PLA2-Ca*
|
| | Leslie and Channon BBA 1045 (1990) 261-270 fig6 pp267. |
| 3 | PLA2*-Ca-act | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PLA2
Pathway No. : 314 | 6
(uM^-1 s^-1) | 0.1
(s^-1) | Kd(bf) = 0.0167(uM) | - | Substrate
Ca
PLA2*
Product
PLA2*-Ca
|
| | To start off, same kinetics as the PLA2-Ca-act direct pathway. Oops ! Missed out the Ca input to this pathway first time round. Let's raise the forward rate about 3x to 5e-6. This will let us reduce the rather high rates we have used for the kenz on PLA2*-Ca. In fact, it may be that the rates are not that different, just that this pathway for getting the PLA2 to the memb is more efficien.... |
| 4 | Act-PLC-Ca | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PLCbeta
Pathway No. : 315 | 3
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 0.3333(uM) | - | Substrate
Ca
PLC
Product
PLC-Ca
|
| | Affinity for Ca = 1uM without AlF, 0.1 with: from Smrcka et al science 251 pp 804-807 1991 so [Ca].kf = kb so kb/kf = 1 * 6e5 = 1/1.66e-6 11 June 1996: Raised affinity to 5e-6 to maintain balance. See notes. |
| 5 | PLC-Gq-bind-Ca | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PLCbeta
Pathway No. : 315 | 29.9997
(uM^-1 s^-1) | 1
(s^-1) | Kd(bf) = 0.0333(uM) | - | Substrate
Ca
PLC-Gq
Product
PLC-Ca-Gq
|
| | this step has a high affinity for Ca, from Smrcka et al. 0.1uM so kf /kb = 1/6e4 = 1.666e-5:1. See the Act-PLC-by-Gq reac. 11 Jun 1996: Raised kf to 5e-5 based on match to conc-eff curves from Smrcka et al. |
| 6 | Ca_act_PLC_g | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PLC_g
Pathway No. : 321 | 180
(uM^-1 s^-1) | 10
(s^-1) | Kd(bf) = 0.0556(uM) | - | Substrate
Ca
PLC_g
Product
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. |
| 7 | Ca_act_PLC_g* | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PLC_g
Pathway No. : 321 | 11.9997
(uM^-1 s^-1) | 10
(s^-1) | Kd(bf) = 0.8334(uM) | - | Substrate
Ca
PLC_G*
Product
Ca.PLC_g*
|
| | Again, we refer to Homma et al and Wahl et al, for preference using Wahl. Half-Max of the phosph form is at 316 nM. Use kb of 10 as this is likely to be pretty fast. Did some curve comparisons, and instead of 316 nM giving a kf of 5.27e-5, we will use 8e-5 for kf. 16 Sep 97. As we are now phosphorylating the Ca-bound form, equils have shifted. kf should now be 2e-5 to match the curves. |
| 8 | CaM-TR2-bind-Ca | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | CaM
Pathway No. : 323 | 72.0001
(uM^-2 s^-1) | 72
(s^-1) | Kd(af) = 1(uM) | - | Substrate
Ca
Ca
CaM
Product
CaM-TR2-Ca2
|
| | Lets use the fast rate consts here. Since the rates are so different, I am not sure whether the order is relevant. These correspond to the TR2C fragment. We use the Martin et al rates here, plus the Drabicowski binding consts. All are scaled by 3X to cell temp. kf = 2e-10 kb = 72 Stemmer & Klee: K1=.9, K2=1.1. Assume 1.0uM for both. kb/kf=3.6e11. If kb=72, kf = 2e-10 (Exactly the same !).... |
| 9 | CaM-TR2-Ca2-bind
-Ca | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | CaM
Pathway No. : 323 | 3.6
(uM^-1 s^-1) | 10
(s^-1) | Kd(bf) = 2.7778(uM) | - | Substrate
Ca
CaM-TR2-Ca2
Product
CaM-Ca3
|
| | K3 = 21.5, K4 = 2.8. Assuming that the K4 step happens first, we get kb/kf = 2.8 uM = 1.68e6 so kf =6e-6 assuming kb = 10 |
| 10 | CaM-Ca3-bind-Ca | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | CaM
Pathway No. : 323 | 0.465
(uM^-1 s^-1) | 10
(s^-1) | Kd(bf) = 21.5052(uM) | - | Substrate
Ca
CaM-Ca3
Product
CaM-Ca4
|
| | Use K3 = 21.5 uM here from Stemmer and Klee table 3. kb/kf =21.5 * 6e5 so kf = 7.75e-7, kb = 10 |
| 11 | Ca-bind-CaNAB-Ca
2 | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PP2B
Pathway No. : 325 | 3.6
(uM^-2 s^-1) | 1
(s^-1) | Kd(af) = 0.527(uM) | - | Substrate
Ca
Ca
CaNAB-Ca2
Product
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 pg 6863, the Kact is 0.5 uM. kf/kb = 1/(0.5 * 6e5)^2 = 1.11e-11 |
| 12 | Ca-bind-CaNAB | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PP2B
Pathway No. : 325 | 10008.4
(uM^-2 s^-1) | 1
(s^-1) | Kd(af) = 0.01(uM) | - | Substrate
Ca
Ca
CaNAB
Product
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, we get kf = 2.78e-8, kb = 1. Try 20 nM. kf = 7e-9, kb = 1 |
| 13 | trigger_PKM | Ajay_Bhalla_
2004_PKM_Tuning
Accession No. : 76 | PKM
Pathway No. : 328 | 0.1
(uM^-2 s^-1) | 0.4
(s^-1) | Kd(af) = 2(uM) | - | Substrate
Ca
Ca
Ca_detector
Product
Ca.detector_
complex
|
color.