EXPERIMENTAL DATA
IN BACTERIAL
CHEMOTAXIS
CARL J MORTON-FIRTH AND ROBERT B BOURRET
12 FEBRUARY 2016
Background
The aim of this document is to provide a source of rate constants and concentrations connected with bacterial chemotaxis in Escherichia coli from published
experimental data. Originally, it was used as a data source for the development of computer models, but has since matured into a resource which may be of use to
other groups. With this in mind, we welcome feedback on these tables and any new data which has not been included, so that we may produce a more accurate
and complete document.
Please send your comments to:
Carl Morton-Firth
Department of Zoology
University of Cambridge
Downing Street
CAMBRIDGE
CB2 3EJ
ENGLAND
E-Mail: cjm18@cam.ac.uk
116103993
Page 1
EXPERIMENTAL DATA IN BACTERIAL CHEMOTAXIS
CONTENTS
Notes ......................................................................................................................................................................................................... 2
Core Reactions ......................................................................................................................................................................................... 4
Complex Formation ...............................................................................................................................................................................................................................4
Aspartate Binding ..................................................................................................................................................................................................................................6
Autophosphorylation..............................................................................................................................................................................................................................7
Phosphotransfer ..................................................................................................................................................................................................................................11
CheY and CheB Phosphorylation ........................................................................................................................................................................................................12
Methylation Reactions ........................................................................................................................................................................... 14
Methylation ..........................................................................................................................................................................................................................................14
Demethylation .....................................................................................................................................................................................................................................16
Aspartate Binding ................................................................................................................................................................................................................................18
Autophosphorylation............................................................................................................................................................................................................................19
Phosphotransfer ..................................................................................................................................................................................................................................21
Double Methylation ..............................................................................................................................................................................................................................22
Other Reactions ..................................................................................................................................................................................... 23
Alternative Reactions for CheY and CheB Phosphorylation ...............................................................................................................................................................23
CheA Dephosphorylation ....................................................................................................................................................................................................................25
Double Autophosphorylation ...............................................................................................................................................................................................................26
CheAS Reactions .................................................................................................................................................................................................................................27
Motor Reactions ..................................................................................................................................................................................................................................28
CheY-CheZ Interactions ......................................................................................................................................................................................................................30
Miscellaneous .....................................................................................................................................................................................................................................31
Protein Concentrations.......................................................................................................................................................................... 33
References .............................................................................................................................................................................................. 35
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 2
116103993
Notes
 Rates in italic type indicate values used by BCT version 3.0. BCT is a computer program which simulates bacterial chemotaxis (Bray et al, 1993; Bray & Bourret,
1995).
 All data should be accurate at 25C.
 All values are quoted in standard units unless otherwise specified (eg M , s-1 , M-1 s-1 , M s-1 ).
 All data comes from experiments using Escherichia coli unless stated otherwise
 The following chemical symbols are used:
A
AS
B
R
T
W
CheA
CheA (short transcript)
CheB
CheR
Tar
CheW
Y
Z
a
m
p
CheY
CheZ
Aspartate
Methyl group
Phosphate
 Rates are quoted for the reaction presented. For example, if a rate, k, is given for the reaction A + B  C + D, the rate of the reaction is k [A] [B].
 Values are quoted to the same accuracy as the least accurate figure used to calculate the value.
 If a rate depends on the concentration of a species which has not been listed as a substrate in the reaction, then the rate quoted assumes this species is at
normal cellular concentrations. This is especially important when a reaction is presented which is a simplification of the real reactions occurring; for instance, the
reaction:
A
 B
k
May be a simplification of:
M
cat
A  x 

 Ax 
B y
K
k
Where the concentration of x is held constant by the cell and [x] » [A] . In this case, the rate constant would be presented as:
k
kcat  x 
K M   x
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 3
 The following constants are used in calculating the reaction rates:
Cell volume
[ATP]cell
[POAc]cell
KM of CheA for ATP
KM of CheY for POAc
1.41 x 10-15 litres
3 x 10-3 M
1 x 10-5 M
3 x 10-4 M
3.2 x 10-3 M
Carl J Morton-Firth and Robert B Bourret
(Kuo & Koshland, 1987)
(Bochner & Ames, 1982)
(Pruss & Wolfe, 1994)
(Wylie et al, 1988; McNally & Matsumura, 1991; Tawa & Stewart, 1994)
(Silversmith et al, 1997) - cf 7 x 10-4 M (Lukat et al, 1992)
12 February 2016
Page 4
116103993
Core Reactions
Complex Formation
Reaction
Rate Constant
kright
TTW
TT + W
3.65 x
kleft
10-3
1.00 x
Source
Comments
3.65 x 10-9
Bray & Bourret, 1995
Derived by rate constant optimisation.
2.0 x 10-5
Surette & Stock, 1996
1.0 x 10-5
Gegner et al, 1992
KD
106
3.65 x 10-9
WAA
 W + AA
8.94 x
10-3
1.00 x
106
8.94 x
10-9
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
Derived by rate constant optimisation.
There is experimental evidence for the existence of this in vivo (McNally & Matsumura, 1991);
however WAA has not been isolated in vitro, but there are theoretical reasons why this might
be the case (Zimmerman & Minton, 1993; Timasheff, 1993).
1.7 x 10-5
8.94 x
TTWAA
 TT + WAA
2.97 x 102
1.00 x 106
2.97 x 10-4
2.97 x
TTWWAA  TTW + WAA
6.40 x 10-1
1.00 x 106
1.12 x 10-1
1.00 x 106
3x
10-6
Derived by rate constant optimisation.
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
10-7
1.12 x 10-7
This was performed at 4C, so for 25C, KD should be higher.
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
10-4
6.40 x 10-7
6.40 x
TTWWAA  TTWW + AA
Gegner & Dahlquist, 1991
10-9
Derived by rate constant optimisation.
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
Derived by rate constant optimisation.
Gegner et al, 1992
This is the dissociation constant of CheA binding Tsr in the presence of CheW. The resultant
complex has a CheW : CheA stoichiometry of 1:1, so this is not the formation of the TTWAA
complex. This figure may not be accurate, as it also reflects:
WWAA  W + WAA  W + W + AA
This involvement of other reactions is suggested by the result that the data indicates 1.5
binding sites of Tsr for CheA.
Note this uses Tsr, not Tar.
1.12 x 10-7
TTWWAA  TT + WWAA
2.29 x
10-2
1.00 x
106
2.29 x
10-8
2 x 10-6
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
Derived by rate constant optimisation.
Gegner et al, 1992
This represents the binding of CheW to Tsr in the presence of CheA, so it reflects a number of
different reactions:
TT+W
TT+WAA
TTW+WAA
TT+WWAA
TTWAA+W
TTW+W
TTAA+W
This is confirmed by the result that the data indicates 1.5 binding sites of Tsr for CheW.
2.29 x 10-8
TTAA
 TT + AA
12 February 2016
3.93 x 101
1.00 x 106
3.93 x 10-5
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
Derived by rate constant optimisation.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 5
Reaction
Rate Constant
kright
kleft
Source
Comments
Gegner et al, 1992
Direct binding of AA to TT in the absence of W has not been observed experimentally (Gegner
et al, 1992). However, there is genetic and biochemical evidence that TT can influence AA
activity in the absence of W (Liu & Parkinson, 1989; Ames & Parkinson, 1994): if Tar is added
to AA in the absence of CheW, autophosphorylation is reduced; overexpression of Tar in W -Zmutant leads to smooth swimming (T-W -Z- has a wild type bias, irrespective of the presence of
stimuli) so it has been suggested that the Tar sequesters the CheA in an inactive TTAA
complex.
KD
> 1.0 x 10-4
3.93 x 10-5
TTWAA
 TTW + AA
7.27 x
102
1.00 x
106
7.27 x 10-4
7.27 x
TTWWAA  TTWAA + W
7.87 x 10-6
1.00 x 106
 TTW + W
5.11 x 10-2
1.00 x 106
 W + WAA
1.02 x 10-1
1.00 x 106
1.7 x
10-5
 TTAA + W
6.76 x 10-2
1.00 x 106
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Derived by rate constant optimisation.
Gegner & Dahlquist, 1991
Scatchard analysis suggest CheW binding to CheA is noncooperative, so this reaction should
have the same KD as WAA  W + AA.
This value is used in BCT (1995), based on Bray & Bourret, 1995.
6.76 x 10-8
Bray & Bourret, 1995
Derived by rate constant optimisation.
0
Bourret, 1996
(From C-11) Analysis of mutant phenotypes suggest that this reaction may not occur.
6.76 x 10-8
Carl J Morton-Firth and Robert B Bourret
Derived by rate constant optimisation.
Bray & Bourret, 1995
1.02 x 10-7
TTWAA
Derived by rate constant optimisation.
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
10-8
1.02 x 10-7
Derived by rate constant optimisation.
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
10-12
5.11 x 10-8
5.11 x
WWAA
10-4
7.87 x 10-12
7.87 x
TTWW
This value is used in BCT (1995), based on Bray & Bourret, 1995.
Bray & Bourret, 1995
This value is used in BCT (1995), based on Bray & Bourret, 1995.
12 February 2016
Page 6
116103993
Aspartate Binding
Reaction
Rate Constant
kright
TT + a
kleft
 TTa
1 x 109
Source
Comments
1.20 x 10-6
Biemann & Koshland, 1994
This is the dissociation constant for free Tar. The dissociation constants in S
typhimurium are measured as 2.0 M and 0.1 M (ie two binding sites). Both
membrane-bound and detergent-soluble receptors give the same results.
1.4 x 10-6 ± 0.5 x 10-6
Danielson et al, 1994
Using S typhimurium.
KD
kright comes from isolated ligand-binding domain. KD comes from a specially
engineered protein.
In principle, conformational changes associated with aspartate binding and release
could be slower in intact transmembrane receptors, where the motion in the
transmembrane and cytoplasmic domains are coupled to aspartate binding.
2 x 10-6
Milligan & Koshland, 1993
Using S typhimurium. Only the periplasmic domain of the receptor was used.
7.8 x 10-7
Dunten & Koshland, 1991
This is the dissociation constant for free Tar.
Bogonez & Koshland, 1985
Using S typhimurium. The receptors are detergent-soluble.
Wang & Koshland, 1980
Using E coli (receptors are membrane bound). Also, the dissociation constant in S
typhimurium is measured as 5 M (receptors are detergent-soluble).
3x
10-6
7 x 10-6
1 x 109
1 x 103
1 x 10-6
This value is used in BCT (1995), based on: Biemann & Koshland, 1994; Danielson
et al, 1994; Dunten & Koshland, 1991.
Only one aspartate binds per Tar dimer (Milburn et al, 1991; Yeh et al, 1993;
Milligan & Koshland, 1993; Biemann & Koshland, 1994).
TTWWAA + a  TTaWWAA
3 x 10-6
Mowbray & Koshland, 1990
Using E coli. The receptors are membrane-bound.
5 x 10-6
Russo & Koshland, 1983
Using S typhimurium. The receptors are membrane-bound.
10-6
Clarke & Koshland, 1979
Using E coli. Also, the dissociation constant in S typhimurium is measured as 5 M
(Figure 2A) and 6 M (Table II). The receptors are membrane-bound.
5x
TTAA + a
 TTaAA
TTW + a
 TTaW
TTWW + a
 TTaWW
TTWAA + a
 TTaWAA
TTAAp + a
 TTaAAp
TTWAAp + a
 TTaWAAp
See TT
BCT assumes that the rate is the same as TT binding.
See TT
BCT assumes that the rate is the same as TT binding.
TTWWAAp + a  TTaWWAAp
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 7
Autophosphorylation
Reaction
Rate Constant
Source
Comments
TTWWAA  TTWWAAp
13.6
Liu et al, 1997
Rate measured in vitro using 50 M excess of CheY and 2.0mM ATP, as 13 s-1.
This is a simplification of the reaction:
TTWWAA + ATP  TTWWAA-ATP  TTWWAAp + ADP
Rate = kcat x [TTWWAA] [ATP] / ( KM + [ATP] )
 kcat = 13 x (2 x 10-3 + 3 x 10-4 ) / (2 x 10-3 )
using KM = 300 M
= 14.95 s-1
This can be converted into a simple first order rate constant using [ATP]cell = 3 x 10-3 M and KD = 300 M.
Note that these rates may refer to an active complex, as described in Liu et al, 1997, with different composition.
Using S typhimurium.
15.5
Stewart, 1993b
> 2.4
Ninfa & Stock, 1991
This is a simplification of the reaction:
TTWWAA + ATP  TTWWAA-ATP  TTWWAAp + ADP
Rate = kcat x [TTWWAA] [ATP] / ( KM + [ATP] )
Where kcat = 17
This can be converted into a simple first order rate constant using K M = 3 x 10-4 M, and [ATP]cell = 3 x 10-3 M.
All the rate constants in this section represent similar simplifications.
CheA autophosphorylation is > 100 x higher in a ternary complex than in isolation; this is based on unpublished data.
Quoted by Stock et al, 1991.
A value of 2.4 x 10-2 is used for CheA autophosphorylation (Tawa & Stewart, 1994).
7.2
Borkovich et al, 1989
CheA autophosphorylation is  300 x higher in an assay containing CheW than without.
A value of 2.4 x 10-2 is used for CheA autophosphorylation (Tawa & Stewart, 1994).
This value is used in BCT (1995), based on Stewart, 1993b.
15.5
kcat = 17 using KM = 3 x 10-4 M, and [ATP]cell = 3 x 10-3 M.
Both CheA can become phosphorylated; only one is phosphorylated in BCT for simplicity (Wolfe & Stewart, 1993;
Swanson et al, 1993; Wolfe et al, 1994).
TTaWWAA  TTaWWAAp
2 x 10-2
Ninfa et al, 1991
(Table 1) Rate of ATP hydrolysis with 0.2 M CheA (limiting), 0.5 M Tar and excess CheY and aspartate is
0.2 M / min = 1 / min
This value may not be comparable to the Stewart, 1993, rate for ternary complex autophosphorylation is the absence
of aspartate due to different reaction conditions.
< 3.87
Borkovich & Simon, 1990
(Figure 4A) CheA-P production in presence of aspartate is 25% of normal. Therefore rate of autophosphorylation is
0.25 x 15.5 (Stewart, 1993)
The rate could be lower because there may be a high concentration of incomplete ternary complexes (eg TTAA) given
the concentrations of T, W and A used.
9.3 x 10-1
0
Carl J Morton-Firth and Robert B Bourret
Borkovich et al, 1989
(Table 2) Maximum CheY-P production in presence of aspartate is 6% of normal. Therefore rate of
autophosphorylation is 0.06 x 15.5 (Stewart, 1993).
This value is used in BCT (1995), based on: Borkovich et al, 1989; Borkovich & Simon, 1990; Ninfa et al, 1991.
12 February 2016
Page 8
116103993
Reaction
AA
 AAp
Rate Constant
Source
Comments
2.1 x 10-1
Liu et al, 1997
Rate measured in vitro using 50 M excess of CheY and 2.0mM ATP, as 0.20s-1.
This is a simplification of the reaction:
CheA + ATP  CheA-ATP  CheA-P + ADP
Rate
= kcat x [A] x [ATP] / ( [ATP] + KD )
 kcat = 0.20 x (2 x 10-3 + 3 x 10-4 ) / (2 x 10-3 ) using KD = 300 M
= 0.23 s-1
This can be converted into a simple first order rate constant using [ATP] cell = 3 x 10-3 M and KD = 300 M.
Using S typhimurium.
1.1 x 10-1
Surette et al, 1996
6 x 10-2
Surette et al, 1996
(Figure 1) This is a simplification of the reaction:
CheA + ATP  CheA-ATP  CheA-P + ADP
Where KD = 274 M; kcat = 1.2 x 10-1 s-1
This can be converted into a simple first order rate constant using [ATP] cell = 3 x 10-3 M.
Using S typhimurium proteins in E coli.
(Figure 6) Performed at pH 8.4, but report a slightly faster rate at pH 7.5, which is contrary to expectations. However,
the reaction at pH 8.5 should be approximately half the speed of the reaction at pH 7.5 according to previous work
(Conley et al, 1994). Different buffer compositions were used in the two measurements, which could explain the
ambiguity.
This is a simplification of the reaction:
CheA + ATP  CheA-ATP  CheA-P + ADP
Where KD = 274 M; kcat = 1.2 x 10-1 s-1
This can be converted into a simple first order rate constant using [ATP]cell = 3 x 10-3 M.
Using S typhimurium proteins in E coli.
Data for the reverse reaction is presented (see Other Reactions: CheA Dephosphorylation)
Because [ATP] =3,000M and [ADP]=250M in vivo, most CheA is ATP-bound. This suggests that regulation of CheA
must be performed by changing kcat, not KD.
4.7 x 10-2
Tawa & Stewart, 1994
Performed at pH 7.5. This is a simplification of the reaction:
CheA + MgATP  CheA-MgATP  CheA-P + ADP
Where KD = 300  75 M; kcat = 5 x 10-2 s-1 (Figure 4)
This can be converted into a simple first order rate constant using [ATP] cell = 3 x 10-3 M.
Note that this is in the presence of CheY and CheZ. In isolation, the k cat was measured at 2.6 x 10-2  0.4 x 10-2 s-1
(Table 1).
Experiments show both CheA are phosphorylated.
Using E coli.
Data for the reverse reaction is presented (see Other Reactions: CheA Dephosphorylation)
12 February 2016
2 x 10-2
Conley et al, 1994
This is measured at pH 7.7; at pH 8.5, rate becomes 4 x 10-2 (maximum rate varying pH). This shows rate is strongly
dependant on pH, so the pH should be supplied whenever this rate is measured.
5.83 x 10-2
Stewart, 1993a
Rate constant is 1.17 x 10-1 at 35C; assuming two fold change to correct to 25C. This was obtained in the presence
of 1% glycerol, which could increase the rate of autophosphorylation (Tawa & Stewart ,1994).
Carl J Morton-Firth and Robert B Bourret
116103993
Reaction
Page 9
Rate Constant
Source
Comments
2 x 10-2
Ninfa et al, 1991
(Table I) Rate of ATP hydrolysis with 0.2 M CheA (limiting), 0.5 M Tar and excess CheY is 0.2 M / min = 1 / min.
Also given are rates for other combinations of Che proteins, however it is not clear which Tar, CheW, CheA complexes
are present (with the concentrations used, a significant quantity of CheA could be sequestered in WAA, WWAA
complexes).
2.5 x 10-2
Lukat et al, 1991
(Figure 3)
3.7 x 10-3
Hess et al, 1988b
(Figure 1) Rate = 425 x 10-12 / 60 mole s-1 mg-1 = 1.03 x 10-3 M s-1 (M CheA)-1 at [ATP] = 100 M
Using MR for CheA dimer = 146,000
From reaction: A + ATP  A-ATP  A-P + ADP
Rate = kcat x [A] x [ATP] / ( [ATP] + KD )
 kcat = 1.03 x 10-3 x (3 x 10-4 + 1 x 10-4 ) / 10-4
using KD = 300 M
= 4.1 x 10-3
This can be converted into a simple first order rate constant using [ATP] cell = 3 x 10-3 M and KD = 300 M.
This is an underestimate of the real value because of the method used (TCA precipitation hydrolyses more than half of
the label off the CheA).
2.27 x 10-2
This value is used in BCT (1995), based on Tawa & Stewart,1994.
kcat = 2.5 x 10-2 using KM = 3 x 10-4 M, and [ATP]cell = 3 x 10-3 M.
WAA
 WAAp
See AA
Surette et al, 1996
The addition of CheW to CheA does not change rate of autophosphorylation.
Using S typhimurium proteins in E coli.
See AA
This value is used in BCT (1995), based on Tawa & Stewart,1994.
This may be higher than the AA value before the ATP concentration is taken into account (after the ATP concentration
is included in the rate constant, the difference should be small because at the high levels of ATP found in the cell, the
difference in KM does not make much difference) explained in detail as follows:
T-Z- and W++T-Z- tumble more than T-W-Z- mutants (Liu & Parkinson, 1989; Ames & Parkinson, 1994), implying CheW
has tumble-generating ability in the absence of Tar, so autophosphorylation of WAA may be faster. WAA has the
same vmax as AA, but a 70-fold lower KM for ATP than AA (McNally & Matsumura, 1991); but [ATP] in the cell is 10
times higher than the KM of AA for ATP (Tawa & Stewart, 1994; Bochner & Ames, 1982), so the difference between
the autophosphorylation rate of WAA and AA will not be large (both are easily saturated with ATP). However, bias is
highly dependent on CheY-P (because of the Hill coefficient) so this could explain the discrepancy observed
experimentally (BCT predicts a difference in bias of 0.1).
WWAA
 WWAAp
See AA
Surette et al, 1996
The addition of CheW to CheA does not change rate of autophosphorylation.
Using S typhimurium proteins in E coli.
See AA
This value is used in BCT (1995), based on Tawa & Stewart,1994.
BCT does not include the fact that WWAA has a much lower K M for ATP than AA (5 M vs. 300 M). However, this
simplification has little consequence at physiological ATP concentration (3 mM), which is essentially saturating for
both reactions (McNally & Matsumura, 1991; Bochner & Ames, 1982).
TTAA
 TTAAp
See AA
This value is used in BCT (1995), based on Tawa & Stewart,1994.
Communication between TT and AA in the absence of W may result in reduced autophosphorylation: if Tar is added to
AA in the absence of CheW, autophosphorylation is reduced; overexpression of Tar in W -Z- mutant leads to smooth
swimming (T-W-Z- has a wild type bias, irrespective of the presence of stimuli) so it has been suggested that the Tar
sequesters the CheA in a TTAA complex (Ames & Parkinson, 1994; Liu & Parkinson, 1989).
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 10
116103993
Reaction
TTWAA
Rate Constant
 TTWAAp
See AA
Source
Comments
This value is used in BCT (1995), based on Tawa & Stewart,1994.
Note that the experimental evidence concerning whether a complex containing only one functional CheW connection
between Tar and CheA has the low activity of AA or the high activity of the TTWWAA ternary complex is contradictory
(Swanson et al, 1993; Wolfe et al, 1994).
TTaAA
 TTaAAp
See AA
2 CheW molecules may be required for transmission of the CCW signal within the signalling complex.
See TTaWWAA
This value is used in BCT (1995), based on Tawa & Stewart,1994.
Note that CheW is not required for transmission of a CCW signal from Tar to CheA (Ames & Parkinson, 1994).
TTaWAA
 TTaWAAp
12 February 2016
See AA
2 CheW molecules may be required for transmission of the CCW signal within the signalling complex.
See TTaWWAA
This value is used in BCT (1995), based on Tawa & Stewart,1994.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 11
Phosphotransfer
Reaction
B + AAp
Rate Constant
 Bp + AA
6x
106
2.08 x 105
>4x
B + WAAp
 Bp + WAA
B + WWAAp
 Bp + WWAA
B + TTAAp
 Bp + TTAA
B + TTWAAp
 Bp + TTWAA
B + TTWWAAp
 Bp + TTWWAA
B + TTaAAp
 Bp + TTaAA
B + TTaWAAp
 Bp + TTaWAA
105
Source
Stewart,
Comments
1993b
Data from stopped-flow kinetics experiments. Earlier (slower) data use curve fitting with coupled NADH
oxidation reactions, which are not particularly sensitive for reactions this fast according to Stewart.
Stewart, 1993a
Rate constant = 4.17 x 105 measured at 35C. To convert this to 25C the rate is halved (Stewart, 1993c ).
Lupas & Stock, 1989
From this, a value of 1 x 106 is used in Bray et al, 1993.
6 x 106
This value is used in BCT (1995), based on Stewart, 1993b.
See AAp
This value is used in BCT (1995), based on Stewart, 1993b; BCT assumes that the rates are the same as
AAp phosphotransfer rates.
B + TTaWWAAp  Bp + TTaWWAA
Y + AAp
 Yp + AA
3 x 107
Stewart, 1993b
Data from stopped-flow kinetics experiments. Earlier (slower) data use curve fitting with coupled NADH
oxidation reactions, which are not particularly sensitive for reactions this fast according to Stewart.
CheY requires Mg2+ for phosphorylation (Lukat et al, 1990; Welch et al, 1994).
2 x 105
3x
Y + TTaWWAAp  Yp + TTaWWAA
Y + WAAp
 Yp + WAA
Y + WWAAp
 Yp + WWAA
Y + TTAAp
 Yp + TTAA
Y + TTWAAp
 Yp + TTWAA
Y + TTWWAAp
 Yp + TTWWAA
Y + TTaAAp
 Yp + TTaAA
Y + TTaWAAp
 Yp + TTaWAA
Lukat et al, 1991
107
See AAp
Performed at 24C.
This value is used in BCT (1995), based on Stewart, 1993b.
Borkovich & Simon, 1990
The rates are the same as the AAp phosphotransfer rate.
See AAp
This value is used in BCT (1995), based on Stewart, 1993b and Borkovich & Simon, 1990
See AAp
This value is used in BCT (1995), based on Stewart, 1993b and Borkovich & Simon, 1990; BCT assumes
that the rates are the same as AAp and TTaWWAAp phosphotransfer rates.
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 12
116103993
CheY and CheB Phosphorylation
Reaction
Y
 Yp
Rate Constant
5.30 x
10-4
Source
Comments
Silversmith et al, 1997
This is a simplification of the reaction Y + POAc  Y-POAc  Yp + OAc.
Rate = kcat x [Y] [POAc] / ( KM + [POAc] )
From Discussion, kcat = 0.17.
This can be converted into a simple first order rate constant using [POAc]cell = 10-5 M and KM = 3.2 x 10-3 M.
Using S typhimurium.
1.24 x 10-3
Lukat et al, 1992
This is a simplification of the reaction Y + POAc  Y-POAc  Yp + OAc.
Rate = kcat x [Y] [POAc] / ( KM + [POAc] )
From figure 4, Rate of acetyl phosphate hydrolysis = 0.083 [Y]
 kcat = 8.8 x 10-2 using [POAc]expt = 10mM and KM = 7 x 10-4 M (Lukat et al, 1992).
This can be converted into a simple first order rate constant using [POAc] cell = 10-5 M and KM = 7 x 10-4 M (Lukat et al, 1992).
CheY overexpression in a Tar deletion mutant gives CW rotation. There are a number of explanations for this behaviour:
- CheY autophosphorylation is faster than current values;
- CheY has partial activity in causing CW rotation (Barak & Eisenbach, 1992 a );
- The kinetics of the CheA-mediated reactions so far determined are incorrect;
- There is another source of phosphate, aside from ATP and CheA-P eg other kinases or small molecular weight
phosphodonors.
CheY requires Mg2+ for phosphorylation (Lukat et al, 1990; Welch et al, 1994).
1.24 x 10-3
This value is used in BCT (1995), based on Lukat et al, 1992.
kcat = 8.8 x 10-2 using KM = 7 x 10-4 M, and [POAc]cell = 10-5 M.
Yp
Y
4.8 x 10-2
3.3 x
10-2
4.5 x 10-2
4x
10-2
Silversmith et al, 1997
Using S typhimurium.
Schuster et al, 1997
Stewart, 1997b
(Figure 1B)
Wang & Matsumura, 1996
Calculated from t1/2 of CheY-P = 210 sec. Performed at 4C, so the rate constant, 3.3 x 10-3 is multiplied by 4, assuming
doubling every 10C.
[Mg2+ ], which is required for dephosphorylation, is 0.1mM in this experiment. The K D for Mg2+ binding to CheY-P is approx
0.2mM (Welch et al, 1994) so at most, only a third of CheY will be bound to Mg 2+. To account for this, the rate constant is
multiplied by 3 (on the basis that most CheY in the cell is bound to Mg2+ ).
This is very approximate.
2.7 x 10-2
3.7 x
10-2
6.9 x 10-2
4.1 x
10-2
1.7 x 10-1
Bren et al, 1996
(Figure 4) Calculated from t1/2 of CheY-P = 26 sec
Lukat et al, 1991
Ninfa & Stock, 1989
Hess et al,
1988a
Hess et al, 1988b
Calculated from t1/2 of CheY-P = 10 sec; this is based on unpublished data. Quoted by Stock et al, 1989.
(Figure 2A) Calculated from t1/2 of CheY-P = 17 sec
(Table 1) CheA-P is in excess, so reaction is limited by CheY-P dephosphorylation, not phosphotransfer from CheA-P to CheY.
Rate of reaction = 1000 nmol min-1 mg-1 = 1 x 10-3 x 14,000 / 60 = 2.3 x 10-1 s-1 (using MR of CheY = 14,000).
This is the upper limit of the rate. This rate should be lower because trichloroacetic acid is used to stop the reaction.
Trichloroacetic acid can partially dephosphorylate CheA-P and CheY-P.
3.7 x 10-2
12 February 2016
This value is used in BCT (1995), based on Lukat et al, 1991.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 13
Reaction
Rate Constant
Source
Comments
Yp + Z  Y + Z
1.61 x 107
Wang & Matsumura, 1996
Calculated from t1/2 of CheY-P = 148 sec and [Z] = 3.5 nM. Performed at 4C, so the rate constant, 1.34 x 106 is multiplied by 4,
assuming doubling every 10C.
[Mg2+ ], which is required for dephosphorylation, is 0.1mM in this experiment. The K D for Mg2+ binding to CheY-P is approx
0.2mM (Welch et al, 1994) so at most, only a third of CheY will be bound to Mg 2+. To account for this, the rate constant is
multiplied by 3 (on the basis that most CheY in the cell is bound to Mg2+ ).
This is very approximate, but it is accurate to say that this rate is 4.05 x 108 times faster than CheY-P auto-dephosphorylation.
CheZ probably exists as a dimer normally (Blat & Eisenbach, 1996a), so these rates should be doubled for the reaction Yp + ZZ.
4.6 x
106
Huang & Stewart, 1993
(Figure 4) Using E coli. Calculated from t1/2 of CheY-P = 7.5 sec with 0.02 M CheZ.
1.5 x
106
Huang & Stewart, 1993
(Figure 5) Using S typhimurium.
Calculated from ATPase activity of 0.1 M CheZ = 133 M/min (assay is CheZ limited at this point)
k = Rate / [Yp] [Z] = 133 / (60 x 1.5 x 10-6 x 0.1) = 1.5 x 106 M-1 s-1
The actual rate may be lower than this because CheY autodephosphorylation is significant under the experimental conditions
used.
3.2 x 105
Lukat et al, 1992
From figure 1, after adding 1.2 mM POAc, amount of CheY is approx same as after adding a further 1.2 mM and 26 nM CheZ.
After 1.2 mM POAc:
 [Y] / [Yp] = 19 kauto / 12 kcat
Rate of CheY-P production = kcat x [Y] [POAc] / ( KM + [POAc] )
= kcat x [Y] x 12 / 19
using KM = 7 x 10-4 M
= Rate of CheY production
= kauto [Yp]
where kauto = Autodephosphlatn rate
After 2.4 mM POAc + 26 nM Z: Rate of CheY-P production = kcat x [Y] [POAc] / ( KM + [POAc] )
= kcat x [Y] x 24 / 31
using KM = 7 x 10-4 M
= Rate of CheY production
= k [Yp] [Z] + kauto [Yp]
where k = Z dephosphorylation rate
 [Y] / [Yp] = 31 (k [Z] + kauto ) / 24 kcat
Equating these two gives: 38 kauto = 31 (k [Z] + kauto )
 k = 7 kauto / 31 [Z]
= 3.2 x 105
5x
105
Lukat et al, 1991
5 x 105
Bp
B
3.5 x
10-1
6.7 x 10-1
Using kauto = 3.7 x 10-2 and CheZ = 26 nM
This value is used in BCT (1995), based on Lukat et al, 1991.
Stewart,
1993a
Stewart et al, 1990
This is given as 0.7 at 35C. To convert this to 25C, this is halved (Stewart, 1993c), giving 0.35.
(Figure 3) CheA-P is in excess, so reaction is limited by CheB-P dephosphorylation, not phosphotransfer from CheA-P to CheB
(Stewart et al, 1990).
Initial rate of decrease is 2% CheA in 1.5 sec. Initial ratio of CheA : CheB = 1 : 50, therefore rate of generation of CheB by
dephosphorylation is 100% CheB in 1.5 sec = 1 / 1.5
1.4 x 10-1
Ninfa & Stock, 1989
Calculated from t1/2 of CheB-P = 5 sec; this is based on unpublished data. Quoted by Stock et al, 1989.
2.8 x 10-1
Hess et al, 1988b
(Table 1) CheA-P is in excess, so reaction is limited by CheB-P dephosphorylation, not phosphotransfer from CheA-P to CheB.
This is the upper limit of the rate. This rate should be lower because trichloroacetic acid is used to stop the reaction.
Trichloroacetic acid can partially dephosphorylate CheA-P and CheB-P.
3.5 x 10-1
Carl J Morton-Firth and Robert B Bourret
An estimate value of 1.0 was proposed in Bray et al, 1993, calculated from the phosphate balance.
12 February 2016
Page 14
116103993
Methylation Reactions
Methylation
Reaction
Rate Constant
Site 1
TTWWAA
 TTmWWAA
TTAA
 TTmAA
TTWAA
 TTmWAA
TTWWAAp
 TTmWWAAp
TTAAp
 TTmAAp
TTWAAp
 TTmWAAp
NB:
Site 2
Site 3
Site 4
Source
Comments
Li et al, 1997
(From Table 1)
Rate of methylation (in absence of CheB) = 0.12 CH3 per receptor per
min using 2 M CheR
Ave
2.36 x 10-4
Using 200 CheR molecules (Simms et al, 1987),
rate = 0.12 x 200 / (6.022045 x 1023 x 1.41 x 10-15 x 2 x 10-6 x 60)
Using Tsr
1.84 x 10-4
1.72 x 10-3
3.94 x 10-3
3.08 x 10-4
1.54 x 10-3
Shapiro et al, 1995
The ratio of methylation rates at site 1 : 2 : 3 : 4 = 30 : 280 : 640 : 50
(Shapiro et al, 1995). The rates are calculated using an average rate of
1.54 x 10-3 s-1 (Simms et al, 1987)
1.60 x 10-4
1.50 x 10-3
4.20 x 10-3
2.96 x 10-4
1.54 x 10-3
Shapiro & Koshland,
1994
The ratio of methylation rates at site 1 : 2 : 3 : 4 = 26 : 244 : 682 : 48
(Shapiro & Koshland, 1994). The rates are calculated using an average
rate of 1.54 x 10-3 s-1 (Simms et al, 1987)
> 5 x 10-3
Lupas & Stock, 1989
In B++ S typhimurium mutants, methyl turnover is only slightly higher than
wild type. This implies the methylation reaction is rate limiting. Maximum
methyl turnover in the B++ mutant = 3200 cell-1 min-1
Using 200 CheR molecules (Simms et al, 1987) and 10,000 molecules of
MCP (Gegner et al, 1992) gives:
v > 3200 / (10,000 x 60) = 5 x 10-3 s-1
CheR concentration is
built into rate constant.
The rates are in vivo so cell concentrations of [AdoMet] applies.
This value is quoted in Stock & Surette,1996, as comparing favourably
with the CheR-catalysed methylation rate from other sources.
1.54 x 10-3
Simms et al, 1987
vmax = 180 (nmol of all MCPs) min-1 (mg enzyme)-1 (Simms et al, 1987)
vmax = 180 x 10-9 x 1,000 x [CheR] x (MR CheR) / { [MCP] x 60 }
Using MR of CheR = 30,000 with 200 CheR molecules (Simms et al,
1987) and 10,000 molecules of MCP (Gegner et al, 1992) gives:
vmax = 1.80 x 10-3 s-1
But if KM = 1.7 x 10-5 (Simms et al, 1987) and [AdoMet] = 100M
(Borczuk et al, 1987),
v = 1.54 x 10-3 inside cell, accounting for [AdoMet]
In reality, assuming [AdoMet] is constant, reactions should be:
CheR + TTWWAA  CheR-TTWWAA  CheR + TTmWWAA
KD = 2.1 x 10-6; KM = 4.2 x 10-6; kcat = 0.11 (Simms et al, 1991)
 kass = 5.24 x 104 M-1s-1; kdis = 0.11 s-1; kcat = 0.11 s-1
However this data implies kcat is not limiting (because not all of the CheR
is bound in the complex).
6.67 x 10-5
12 February 2016
2.50 x 10-4
8.33 x 10-4
1.67 x 10-5
2.92 x 10-4
Terwilliger et al,
1986
These give ratio of rates for site 1 : 2 : 3 : 4 = 57 : 214 : 714 : 14
The rates are in vivo so cell concentrations of [AdoMet] applies.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 15
Reaction
Rate Constant
Site 1
TTaWWAA
 TTmaWWAA
TTaAA
 TTmaAA
TTaWAA
 TTmaWAA
Site 2
Site 3
Site 4
Source
Comments
Le Moual et al,
1997; Simms et al,
1987
Using the rate of methylation for non-aspartate bound receptor
complexes from Simms et al, 1987; using increase in methylation rate
from Le Moual et al, 1997.
 Rate for asp bound receptor = (1.54 x 10-3) x 1.43
= 2.20 x 10-3
Ave
2.20 x 10-3
TTaWWAAp 
TTmaWWAAp
TTaAAp
 TTmaAAp
TTaWAAp
 TTmaWAAp
NB:
Note that in the absence of the pentapeptide on the receptor which binds
CheR, the rate of methylation drops rapidly, implying the binding of CheR
to the pentapeptide is necessary for methylation by CheR.
3.91 x 10-4
CheR concentration is
built into rate constant.
Li et al, 1997
(From Table 1)
Rate of methylation (in absence of CheB) is 1.66 times rate in absence of
asparate.
Using Tsr
1.05 x
10-2
Simms et al, 1987;
Terwilliger et al,
1986
Using the rate of methylation for non-aspartate bound receptor
complexes from Simms et al, 1987; using increase in methylation rate
from Terwilliger et al, 1986.
 Rate for asp bound receptor = (1.54 x 10-3) x (2 x 10-3) / (2.92 x 10-4)
= 1.05 x 10-2
1.00 x 10-3
3.67 x 10-3
Carl J Morton-Firth and Robert B Bourret
2.83 x 10-3
5.00 x 10-4
2.00 x 10-3
Terwilliger et al,
1986
These give ratio of rates for site 1 : 2 : 3 : 4 = 125 : 459 : 354 : 62
The rates are in vivo so cell concentrations of [AdoMet] applies.
12 February 2016
Page 16
116103993
Demethylation
Reaction
Rate Constant
Site 1
Site 2
Site 3
Site 4
Source
Comments
Lupas & Stock, 1989
Ave
TTmWWAA + CheB
 TTWWAA + CheB
TTmAA + CheB
 TTAA + CheB
Data given as 14% Tar esters hydrolysed per min
per M CheB. Converted to rate constant by:
TTmWAA + CheB
 TTWAA + CheB
No of Tar hydrolysed, T = T0 e -k [B] t
TTmWWAAp + CheB
 TTWWAAp + CheB
TTmAAp + CheB
 TTAAp + CheB
TTmWAAp + CheB
 TTWAAp + CheB
2.51 x 103
 k = - ln (0.86) / (60 x 10-6)
103
Lupas & Stock, 1989
Data given as 1,000 demethylation events per min
in CheA mutant (ie all CheB is unphosphorylated).
6.26 x 102
Terwilliger et al, 1986
These give ratio of rates for site 1 : 2 : 3 : 4 =
1 : 1 : 2.3 : 0.7
1.3 x 102
Springer & Zanolari,
1984
t1/2 of methyl groups in cell (ie all MCPs) in CheA
mutant strain (where all CheB will be
unphosphorylated) is 40 min.
1.2 x 102
Kehry et al, 1985
1.98 x
5.00 x 102
5.00 x 102
1.17 x 103
3.33 x 102
Using [CheB] = 2.27 M (Simms et al, 1985)
TTmaWWAA + CheB
 TTaWWAA + CheB
TTmaAA + CheB
 TTaAA + CheB
TTmaWAA + CheB
 TTaWAA + CheB
TTmaWWAAp + CheB
 TTaWWAAp + CheB
TTmaAAp + CheB
 TTaAAp + CheB
TTmaWAAp + CheB
 TTaWAAp + CheB
TTmWWAA + CheB-P
 TTWWAA + CheB-P
TTmAA + CheB-P
 TTAA + CheB-P
TTmWAA + CheB-P
 TTWAA + CheB-P
Using [CheB] = 2.27 M (Simms et al, 1985)
2.51 x 104
Lupas & Stock, 1989;
Stewart et al, 1990
3.01 x 104
Lupas & Stock, 1989
TTmWWAAp + CheB-P  TTWWAAp + CheB-P
TTmAAp + CheB-P
 TTAAp + CheB-P
TTmWAAp + CheB-P
 TTWAAp + CheB-P
12 February 2016
After addition of excess aspartate, rate of labelled
methanol formation (in pulse-chase experiment)
= 2.69 x 10-4
The ratio of the maximal to minimal rate of
methylesterification is 4-5 : 0.5-0.6  10 for
different levels of CheB phosphorylation (Stewart
et al, 1990). These constants are calculated by
multiplying the constants for non-phosphorylated
CheB by 10 (Lupas & Stock, 1989).
The increase in activity when CheB is
phosphorylated could be as high as 100x because
t1/2 of CheB-P is low, so it is possible that the
increased activity is attributable to only a small
fraction of the CheB molecules (Bourret, 1996;
Stewartc, 1993).
The ratio of demethylation of
site 1 : 2 : 3 : 4 = 1 : 1 : 2.3 : 0.7
based on unphosphorylated CheB demethylation
(Terwilliger et al, 1986).
This represents a 12 x increase in demethylation
when CheB is phosphorylated.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 17
Reaction
Rate Constant
Site 1
TTmaWWAA + CheB-P  TTaWWAA + CheB-P
TTmaAA + CheB-P
 TTaAA + CheB-P
TTmaWAA + CheB-P
 TTaWAA + CheB-P
TTmaWWAAp + CheB-P  TTaWWAAp + CheB-P
TTmaAAp + CheB-P
 TTaAAp + CheB-P
TTmaWAAp + CheB-P
 TTaWAAp
Carl J Morton-Firth and Robert B Bourret
Site 2
Site 3
Site 4
Source
Comments
> 6 x 103
Springer & Zanolari,
1984
Maximum demethylation rate (which occurred after
exposure to chemorepellents) in cells (ie all
MCPs) is > 25 fold higher than unstimulated state
(where t1/2 of methyl groups is 40 min).
7.53 x 103
Borczuk et al, 1986
Methylation rate measured using N-terminal
deleted mutant form of CheB (lacks regulatory
domain; has the same activity as CheBp, but
cannot be controlled - Lupas & Stock, 1989).
Addition of excess aspartate leads to 70% fall in
CheB activity.
Ave
Rate of demethylation of asp-bound receptor
= 0.3 x rate of demethylation of unbound receptor
= 0.3 x 2.51 x 104
(Lupas & Stock, 1989; Stewart et al, 1990).
12 February 2016
Page 18
116103993
Aspartate Binding
Reaction
Equilibrium Constant
TTmWWAA + a  TTmaWWAA
TTmAA + a
 TTmaAA
TTmWAA + a
 TTmaWAA
0 methyl
1 methyl
2 methyl
3 methyl
4 methyl
6.0 x 10-7
9.0 x 10-7
1.20 x 10-6
2.70 x 10-6
4.20 x 10-6
Source
Comments
Borkovich et al,
1992
Ratio of dissociation constants of
EEEE : QEQE : QEmQEm = 1 : 2 : 7 (Borkovich et al, 1992)
by linear interpolation, ratio of constants with varying number of methyls
0 : 1 : 2 : 3 : 4 = 1 : 1.5 : 2 : 4.5 : 7
TTmWWAAp + a  TTmaWWAAp
TTmAAp + a
 TTmaAAp
TTmWAAp + a
 TTmaWAAp
Using dissociation constant of 1.2 M (Biemann & Koshland, 1994) for
wild type receptor. In absence of stimuli, wild type receptor is methylated
twice (Stock & Koshland, 1981), so the '2 methyl' rate constant is fixed
and the other rates adjusted based on the ratios above.
The data may not be valid because they were obtained using isolated
Tar, not the ternary complex (Stock & Surette, 1996).
See also TTWWAA + a  TTaWWAA
Whether or not methylation has an effect on ligand binding could depend
on: location of receptor (free or membrane bound receptor); interactions
of receptor (bound to ternary complex). This could explain the
differences reported in the literature (Iwama et al, 1997).
This data is from E coli
9.4 x
10-7
1.07 x
10-6
1.20 x
10-6
1.32 x
10-6
1.45 x
10-6
Dunten &
Koshland, 1991
Ratio of dissociation constants of
EEEE : QQQQ = 1 : 1.54
by linear interpolation, ratio of constants with varying number of methyls
0 : 1 : 2 : 3 : 4 = 1 : 1.13 : 1.27 : 1.40 : 1.54
Using dissociation constant of 1.2 M (Biemann & Koshland, 1994) for
wild type receptor. In absence of stimuli, wild type receptor is methylated
twice (Stock & Koshland, 1981), so the '2 methyl' rate constant is fixed
and the other rates adjusted based on the ratios above.
This data is from S typhimurium
2.1 x
10-7
7.1 x
10-7
1.20 x
10-6
1.69 x
10-6
2.18 x
10-6
Yonekawa &
Hayashi, 1986
Ratio of [aspartate] for 50% Tar saturation
cheR mutant (EEEE) : cheB mutant (QEmQEm) = 1 : 10
(Ratio is 1 : 100 with Tsr)
by linear interpolation, ratio of constants with varying number of methyls
0 : 1 : 2 : 3 : 4 = 1 : 3.25 : 5.5 : 7.75 : 10
Using dissociation constant of 1.2 M (Biemann & Koshland, 1994) for
wild type receptor. In absence of stimuli, wild type receptor is methylated
twice (Stock & Koshland, 1981), so the '2 methyl' rate constant is fixed
and the other rates adjusted based on the ratios above.
This data is from E coli
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 19
Autophosphorylation
Reaction
TTmWWAA
Rate Constant
 TTmWWAAp
0 methyl
1 methyl
2 methyl
3 methyl
4 methyl
0
6.80 x 100
1.36 x 101
1.72 x 101
2.09 x 101
Source
Comments
Liu et al, 1997
Activity of EEEE : QEQE : QQQQ = 0 : 13 : 20 s -1
By linear interpolation, ratio of constants with varying numbers of
methyls:
0 : 1 : 2 : 3 : 4 = 0 : 6.5 : 13 : 16.5 : 20 s -1
Using conversion described under TTWWAA  TTWWAAp
reaction, to account for different ATP concentrations.
Note that these rates may refer to an active complex, as
described in Liu et al, 1997, with different composition.
1.01 x 10-1
7.80 x 100
1.55 x 101
2.36 x 101
3.17 x 101
Borkovich et al, 1992
Ratio of CheY-P production without asp after 5 sec
Control : EEEE : QEQE : QEmQEm = 700 : 800 : 16,000 :
32,000
By linear interpolation, ratio of constants with varying numbers of
methyls (subtracting control values):
0 : 1 : 2 : 3 : 4 = 6.54 x 10-3 : 0.503 : 1 : 1.523 : 2.046
[This is confirmed by a second set of measurements:
Ratio of IC50 (amount of asp for 50% CheY phosphorylation):
EEEE : QEmQEm = 1 : 185
Taking into account 7x change in ligand binding (Borkovich et al,
1992), by linear interpolation, ratio of constants with varying
number of methyls
0 : 1 : 2 : 3 : 4 = 1 : 6.6 : 13.2 : 19.8 : 26.4]
Using rate constant of 15.5 s-1 (Stewart, 1993b) for wild type
receptor. In absence of stimuli, wild type receptor is methylated
twice (Stock & Koshland, 1981), so the '2 methyl' rate constant
is fixed and the other rates adjusted based on the ratios above.
See also TTWWAA  TTWWAAp
TTmAA
 TTmAAp
TTmWAA
 TTmWAAp
TTmaAA
 TTmaAAp
TTmaWAA
 TTmaWAAp
See TTAA, TTWAA
It is assumed that methylation does not affect rate of
autophosphorylation because there is unlikely to be any means
of conformational signalling between Tar and CheA in these
complexes (only the TTWWAA complex has active Tar to CheA
signalling). However CheW is not required for transmission of
signal from Tar to CheA after aspartate binding (Ames &
Parkinson, 1994).
See TTaAA, TTaWAA
It is assumed that methylation does not affect rate of
autophosphorylation because there is unlikely to be any means
of conformational signalling between Tar and CheA in these
complexes (only the TTWWAA complex has active Tar to CheA
signalling). However CheW is not required for transmission of
signal from Tar to CheA after aspartate binding (Ames &
Parkinson, 1994).
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 20
116103993
Reaction
Rate Constant
0 methyl
TTmaWWAA
 TTmaWWAAp
12 February 2016
6.08 x
10-3
1 methyl
4.68 x
10-1
2 methyl
9.3 x
10-1
3 methyl
1.42 x
100
Source
Comments
Borkovich et al, 1989
(Table 2) Maximum CheY-P production in presence of aspartate
is 6% of normal. Therefore rate of autophosphorylation is 0.06 x
15.5 (Stewart, 1993). This is assumed to apply to complexes in
the second methylation state (complexes are in this state in the
absence of aspartate, Stock & Koshland, 1981).
Using Ratio of CheY-P production from Borkovich et al, 1992, of:
6.54 x 10-3 : 0.503 : 1 : 1.523 : 2.046.
4 methyl
1.90 x 100
Carl J Morton-Firth and Robert B Bourret
116103993
Page 21
Phosphotransfer
Reaction
Rate Constant
B + TTmWWAAp
 Bp + TTmWWAA
B + TTmAAp
 Bp + TTmAA
B + TTmWAAp
 Bp + TTmWAA
B + TTmaAAp
 Bp + TTmaAA
B + TTmaWAAp
 Bp + TTmaWAA
See TTWWAAp
Source
Comments
It is assumed that methylation does not affect rate of phosphotransfer.
B + TTmaWWAAp  Bp + TTmaWWAA
Y + TTmWWAAp
 Yp + TTmWWAA
Y + TTmAAp
 Yp + TTmAA
Y + TTmWAAp
 Yp + TTmWAA
Y + TTmaAAp
 Yp + TTmaAA
Y + TTmaWAAp
 Yp + TTmaWAA
See TTWWAAp
Y + TTmaWWAAp  Yp + TTmaWWAA
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 22
116103993
Double Methylation
Reaction
Rate Constant
TTmWWAA
 TmTmWWAA
TTmAA
 TmTmAA
TTmWAA
 TmTmWAA
TTmWWAAp
 TmTmWWAAp
TTmAAp
 TmTmAAp
TTmWAAp
 TmTmWAAp
TTmaWWAA
 TmTmaWWAA
TTmaAA
 TmTmaAA
TTmaWAA
 TmTmaWAA
See rates for single methylation
Source
Comments
If these reactions are added to the model, the corresponding autophosphorylation,
phosphotransfer, auto-dephosphorylation and demethylation reactions need to be
added for the new, doubly methylated species.
Also, if methylation at every site is implemented, there must be species representing
every combination of two multiple-methylated receptors.
TTmaWWAAp  TmTmaWWAAp
TTmaAAp
 TmTmaAAp
TTmaWAAp
 TmTmaWAAp
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 23
Other Reactions
Alternative Reactions for CheY and CheB Phosphorylation
Reaction
Rate Constant
Source
Comments
KD = 1.5 x 10-6  6 x 10-7
Stewart, 1997a
This suggests a role for free cytoplasmic CheA binding and CheY, preventing it from interacting with
ternary complexes. For example if 50% of CheA is not in a ternary complex, ratio of free [Y] : [A-Y] = 3
ie concentration of CheY is effectively reduced by 25%.
KD = 1.2 x 10-6  8 x 10-8
Shukla &
Matsumura, 1995
Experiment performed at 298 K using fluorescence quenching.
KD = 2.0 x 10-6
Li et al, 1995
Experiment performed at 298 K using ITC (isothermal titration calorimetry).
kright
A-Y  A + Y
kleft
Using first 233 amino acids of CheA instead of complete CheA gives K D = 1.2 x 10-6 (ie no significant
change).
It is suggested that differences between this value and Schuster et al,1993, are due to differences in
buffer composition and peculiarities of the SPR method.
1.14 x 10-5
3.68 x 102
Schuster et al,
1993
KD = 3.10 x 10-8
Used in Hauri & Ross, 1995.
Experiment performed using SPR (surface plasmon resonance).
Note that the reactions investigated by Schuster (AY  A + Y; TTWWAA-Y + ATP  TTWWAA + Yp)
have been compared to the conventional reactions where no association of Y and TTWWAA occurs
(TTWWAA  TTWWAAp; TTWWAAp + Y  TTWWAA + Yp): the basic relationships between velocity
and [Y] are identical, but the two sets of rate constants are not equivalent, and there are significant
discrepancies. Hauri & Ross point out that the rates must be much higher than those given because it
seems unlikely that these constants could lead to the observed behaviour.
The implementation of an association reaction between Y and TTWWAA could lead to a lower effective
concentration of Y in the cell. With these constants, almost all the TTWWAA is bound to Y, and Y is
lowered by approx 10%. These reactions could increase the effect of other, inactive, CheA-containing
complexes, which can bind Y, preventing it binding the active CheA-containing complexes.
Similar reactions are proposed for CheB in Hauri & Ross, 1995, but there is no data available for the
rate constants. The data Hauri & Ross used were actually for different reactions (they are the constants
for the normal autophosphorylation / phosphotransfer reactions !).
KD = 3.7 x 10-7
A-Yp
 A + Yp
KD = 4.0 x 10-6
Ap-Y
 A-Yp
650  200
Ap-Y
 Ap + Y
KD = 6 x 10-6  2 x 10-6
Carl J Morton-Firth and Robert B Bourret
< 50
Swanson et al,
1993
Experiment performed using SPR (surface plasmon resonance).
Li et al, 1995
6mM Mg2+ reduces the binding by a further 3-fold
Stewart,
1997a
The data is actually from a CheA construct containing amino acids 1 - 233, which according to the
research, contains the CheY binding domain (also see Li et al, 1995).
Reaction kinetics are complicated by the fact that a residual level of phosphorylated CheA always
remains even when CheY is in excess.
Stewart, 1997a
12 February 2016
Page 24
116103993
Reaction
Rate Constant
kright
TTWWAA-Y + ATP  TTWWAA + Yp
6.36 x
Source
Comments
Schuster et al,
1993
Used in Hauri & Ross, 1995
kleft
10-2
Scaled up by a factor of six because in original data, [ATP] = 0.5mM, but in cell, [ATP] = 3mM.
kleft could not be determined because the exact concentration of Tar was not known.
TTWWAA-Y
 TTWWAA + Y
A-B
B+A
12 February 2016
1.44 x
10-4
KD = 3.2 x
10-6
Schuster et al,
1993
This experiment is performed in the absence of ATP.
Li et al, 1995
CheB binds CheA in competition with CheY.
kleft could not be determined because the exact concentration of Tar was not known.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 25
CheA Dephosphorylation
Reaction
Ap
Rate Constant
A
6.1 x
10-2
Source
Comments
Surette et al, 1996
Performed at pH 8.4. This is a simplification of the reaction:
CheA-P + ADP  CheA-P-ADP  CheA + ATP
Rate = kcat x [CheA-P] [ADP] / ( KD + [ADP] )
Where KD = 240  52 M; kcat = 1.2 x 10-1 s-1
This can be converted into a simple first order rate constant using [ADP] cell = 250 M.
(Ratio of CheA-P : CheA-P-ADP = 0.5).
Data is also presented for the equilibria
A-ADP  A + ADP
KD = 160  59 M
Ratio of A : A-ADP = 2 : 3
Ap-ATP  Ap + ATP
KD = 810  140 M
Ratio of Ap : Ap-ATP = 1 : 4
These binding reactions could potentially reduce the effective concentration of CheA available for other reactions.
Using S typhimurium proteins in E coli.
The rate of the autodephosphorylation reaction, Ap  A, in the absence of ADP, is relatively insignificant, with a
maximum rate of 1.2 x 10-5 s-1 (Silversmith, 1996).
2.4 x 10-2
Tawa & Stewart, 1994
Performed at pH 7.5. This is a simplification of the reaction:
CheA-P + ADP  CheA-P-ADP  CheA + ATP
Rate = kcat x [CheA-P] [ADP] / ( KD + [ADP] )
Where KD = 42  8 M; kcat = 2.8 x 10-2  0.3 x 10-2 s-1
This can be converted into a simple first order rate constant using [ADP] cell = 250 M.
(Ratio of CheA-P : CheA-P-ADP = 0.17 so most CheA-P is bound to ADP).
AAp
 AA
WAAp
 WAA
WWAAp
 WWAA
TTAAp
 TTAA
TTWAAp
 TTWAA
TTaAAp
 TTaAA
TTaWAAp
 TTaWAA
TTWWAAp
 TTWWAA
See Ap
It is assumed that these rates are the same as for CheA dephosphorylation, but this may not be valid for some or all
of these complexes.
TTaWWAAp  TTaWWAA
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 26
116103993
Double Autophosphorylation
Reaction
TTWWAAp
 TTWWApAp
TTaWWAAp  TTaWWApAp
AAp
 ApAp
WAAp
 WApAp
WWAAp
 WWApAp
TTAAp
 TTApAp
TTWAAp
 TTWApAp
TTaAAp
 TTaApAp
TTaWAAp
 TTaWApAp
12 February 2016
Rate Constant
Source
Comments
See normal
autophosphorylation
reactions
Swanson et al, 1993;
Hess et al, 1987
These reactions are unimportant because the single autophosphorylation rate actually represents net
autophosphorylation rate. Assuming the complex contains two independently autophosphorylated CheA, if two
sites were used, the following reactions would be added:
Phosphorylation of first A:
TTWWAA  TTWWApA
Rate = k/2
TTWWAAp  TTWWApAp
Rate = k/2
Phosphorylation of second A: TTWWAA  TTWWAAp
Rate = k/2
TTWWApA  TTWWApAp
Rate = k/2
Where k is the single autophosphorylation rate.
These can be reduced to the single autophosphorylation equation:
TTWWAA  TTWWAAp
Rate = k
If these reactions are added to the model, the corresponding phosphotransfer, auto-dephosphorylation,
methylation and demethylation reactions need to be added for the new, doubly phosphorylated species.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 27
CheAS Reactions
Reaction
TTWWASA
 TTWWASAp
TTaWWASA  TTaWWASAp
ASA
 ASAp
WASA
 WASAp
WWASA
 WWASAp
TTASA
 TTASAp
TTWASA
 TTWASAp
TTaASA
 TTaASAp
TTaWASA
 TTaWASAp
AS + Z
 AS-Z
AS-Z + Yp
 AS-Z + Y
Rate Constant
Source
Comments
See normal
autophosphorylation
reactions
Wolfe & Stewart, 1993
If CheAS is added to the system, the corresponding binding (complex formation), phosphotransfer, autodephosphorylation (single and double), methylation, demethylation and ligand-binding reactions need to be
added for the new, CheAS-containing species.
Not determined
Wang & Matsumura, 1996
Furthermore, ASAS dimer containing complexes need to be added. These cannot undergo
autophosphorylation, but will reduce the effective concentrations of Tar, CheW and any other chemotactic
proteins that may bind CheA (possibly CheR, CheB and CheY).
CheAS reactions may not be important because deletion of CheA S have wild type phenotypes (Sanatinia et al,
1995). However, co-expression of CheAS and CheZ appears to be limited to motile, chemotactic enteric
bacteria (McNamara & Wolfe, 1997). This suggests there is some important interaction between CheA S and
CheZ; CheAS may be important under certain conditions, which do not normally exist in laboratory
experiments, such as coupling different signalling systems together. For instance, E coli lacking CheA S have a
competitive advantage over wild type cells in semi-solid agar assays; this advantage is enhanced under
anaerobic conditions (McNamara et al, 1997).
The ratio of AS : Z in the AS-Z multimer is 1 : 5.3. The KD for the complex has not been determined.
See comments above for the CheAS autophosphorylation reactions.
2.24 x
108
Wang & Matsumura, 1996
In an experiment performed to determine the stoichiometry of the CheA S-CheZ complex, 1 pmol of CheAS was
incubated with 20 pmol of CheZ. Of this, 0.63 pmol of CheA S and 3.3 pmol of CheZ formed a complex. In the
CheY-P dephosphorylation time course experiment, concentrations of 1.4 nM CheA S and 3.5 nM CheZ were
used. To a first approximation, the 1.4 x 0.63 / 1 nM CheAS and 3.5 x 3.3 / 20 CheZ will be in the complex. The
smaller of these will represent the AS-Z complex concentration = 0.58 nM (CheZ-limiting).
Calculated from t1/2 of CheY-P = 64 sec. Performed at 4C, so the rate constant, 1.87 x 107 is multiplied by 4,
assuming doubling every 10C.
[Mg2+ ], which is required for dephosphorylation, is 0.1mM in this experiment. The KD for Mg2+ binding to
CheY-P is approx 0.2mM (Welch et al, 1994) so at most, only a third of CheY will be bound to Mg 2+. To
account for this, the rate constant is multiplied by 3 (on the basis that most CheY in the cell is bound to Mg 2+ ).
This is very approximate, but it is accurate to say that this rate is 14.0 times faster than normal CheZ (see
Yp + Z dephosphorylation reaction).
It has been suggested that CheAS could provide a direct link between the receptors and CheZ activity because
CheAS can form both Tar-containing complexes and CheZ-containing complexes. It is possible that these
could be linked in some way.
See comments above for the CheAS autophosphorylation reactions.
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 28
116103993
Motor Reactions
Reaction
Yp
Rate Constant
 CW rotation
Bias 
1
 Yp 

1  73 
 Yp0 
Yp + Fumarate  CW rotation
Y
 CW rotation
1% that of CheY-P
Source
Comments
Kuo & Koshland, 1989
Yp0 = Concentration of CheY-P for wild type bias (0.7)
Barak & Eisenbach,
1992b ; Barak et al,
1996
The only research suggesting the existence of a cytoplasmic factor necessary for switching in E coli comes
from research performed by Eisenbach. Fumarate is however used as a switching factor in Halobacteria
(Marwan et al, 1990). Fumarate, probably the cytoplasmic factor, is only required for switching the direction of
the motor, not simply driving the motor in a particular direction.
Barak & Eisenbach,
1992a
Either CheY autophosphorylation is faster than current values, or CheY has partial activity in causing CW
rotation. In the experiments, [CheY] = 36 M, but in vivo, [CheY] = 8 M; therefore this may not be relevant in
vivo.
5.5
The rate constant comes from the fact that when CheY is inserted into semi-lysed cells in phosphorylating
conditions, the switching activity increases 3 fold, and it is found that 2.3% of the CheY are phosphorylated.
Yp-FliM
 Yp + FliM
KD = 1.43 x 10-6
Clegg & Koshland,
1984; Ravid et al, 1986;
Kuo & Koshland, 1987;
Wolfe et al, 1987; Smith
et al, 1988; Conley at al,
1989
CW rotation observed in the absence of CheA and other cytoplasmic Che proteins required for CheY
phosphorylation.
Welch et al, 1994
(From figure 5)
Initial concentration of FliM = 5 n mol in 250 l
Initial concentration of CheY = 3.5 n mol in 250 l
FliM bound
= 0.85 x Initial CheY concentration
Mg2+ is not required for CheY-P to FliM binding, though it is required for CheY phosphorylation.
KD = 1.28 x 10-4
Welch et al, 1993
(From figure 4; the data from figure 3 does not yield sensible values)
Initial concentration of FliM = 3 n mol in 250 l
Initial concentration of CheY = 27 g in 250 l (MR = 14,000)
FliM bound
= 1700 dpm (activity 10.8 dpm / pmol)
Not all the CheY is phosphorylated, so the KD may be lower than this, representing stronger binding.
It is not clear whether FliM and CheZ compete for CheY-P binding, which would be the case if they both bound
at the same site on CheY-P (Blat & Eisenbach, 1994).
Y-FliM
 Y + FliM
KD = 4.43 x 10-4
Welch et al, 1994
(From figure 5)
Initial concentration of FliM = 5 n mol in 250 l
Initial concentration of CheY = 3.5 n mol in 250 l
FliM bound
= 0.042 x Initial CheY concentration
ie CheY binds FliM 310 times more strongly when phosphorylated.
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 29
Reaction
Rate Constant
Source
Comments
KD = 2.48 x 10-3
Welch et al, 1993
(From figure 4; the data from figure 3 does not yield sensible values)
Initial concentration of FliM = 3 n mol in 250 l
Initial concentration of CheY = 27 g in 250 l (MR = 14,000)
FliM bound
= 100 dpm (activity 10.8 dpm / pmol)
ie CheY binds FliM 20 times more strongly when phosphorylated.
Z + FliG / M / N  ?
Yamaguchi et al, 1986;
Parkinson et al, 1983;
Parkinson et al, 1979
Many FliG / FliM / FliN mutations suppress CheZ missense mutations, suggesting an interaction between
CheZ and the motor proteins. This could provide a mechanism to reduce excessively high levels of CheY-P,
and might contribute a non-linear element in the model which could lead to a periodic run-tumble pattern of
swimming behaviour.
Yp-FliM
 Y-FliM
See Yp  Y
Bren et al, 1996
Rate of CheY-P autodephosphorylation is unaffected by binding to FliM.
Yp-FliM + Z
 Y-FliM + Z
0
Bren et al, 1996
CheY-P bound to FliM is protected from CheZ-catalysed dephosphorylation. This is supported by the finding
that CheY mutations which prevent dephosphorylation by CheZ map near to the FliM binding domain;
presumably the CheY-P binds the FliM irreversibly (Sanna et al, 1995).
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 30
116103993
CheY-CheZ Interactions
Reaction
Yp-Z
Rate Constant
 Yp + Z
KD = 5.0 x
10-6
Source
Blat & Eisenbach,
Comments
1996a
(From figure 3B)
KD = [Yp] [Z] / [Yp-Z] = (x-by) (a - by) / by
where x = [Yp], y = Rel band intensity, a = [CheZ]0 , b = [Yp-Z] when rel band int = 0
By finding optimal values for b and KD which minimise the total error for the five points from the dissociation
constant equation, values for KD can be found: KD = 4.96 x 10-6 , b = 1.1 x 10-6
CheZ normally exists as a dimer. In the presence of CheY-P, it oligomerises into a complex containing CheZ
and CheY-P in the ratio 2:1; the size of the oligomer is approximately 4 - 5 times larger than the CheZ dimer,
so it may be the complex: Z-Yp-Z-Z-Yp-Z-Z-Yp-Z-Z-Yp-Z, or Z-Z-Z-Z-Z-Z-Z-Z with 4 associated Yp.
This Yp-Z complex is different from the CheZ-CheAS complex; the former is less stable and dissociates in
the absence of CheY-P.
The oligomerisation step might be very slow, in which case it would allow methylation-independent
adaptation.
KD = 3.98 x 10-6
Blat & Eisenbach, 1994
Graphs of log(Zbound / Yfree) vs log(Zfree) are presented.
KD = Zfree x Yfree / Zbound
Therefore graph is: y = x - KD
This figure is with 2.7mM Mg2+; without Mg2+, KD = 1.58 x 10-5 M (4 fold weaker binding).
There may be multiple CheZ in each complex; under optimal conditions, as many as 12 molecules of CheZ
are bound to each CheY-P.
Y-Z
Y+Z
KD  
Blat & Eisenbach, 1996a
No oligomerisation of CheZ observed if CheY is not phosphorylated.
KD = 6.31 x 10-4
Blat & Eisenbach, 1994
This means CheZ binds CheY  150 x more strongly if CheY is phosphorylated.
Graphs of log(Zbound / Yfree) vs log(Zfree) are presented.
KD = Zfree x Yfree / Zbound
Therefore graph is: y = x - KD
This figure is with 2.7mM Mg2+; without Mg2+, KD = 3.98 x 10-3 M (6.3 fold weaker binding).
Yp-Z* + Yp  Yp-Z* + Y
Z + Yp
Z+Y
12 February 2016
See CheZ catalysed
dephosphorylation of
CheY-P above
Blat & Eisenbach, 1996b
The Yp-Z complex is much more active than the Y-Z complex
Approx 8 times less active
than Yp-Z catalysis
Blat & Eisenbach, 1996b
Three CheZ mutants which do not demonstrate oligomerisation with CheY-P have 5, 9 and 10-fold lower
phosphatase activities than wild type CheZ which can oligomerise.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 31
Miscellaneous
Reaction
AA
Rate Constant
A+A
KD = 1 x
Source
10-7
Stewart,
KD = 2 x 10-7 to 4 x 10-7
Comments
1997a
Surette et al, 1996
Using E coli.
In isolation, this would lead to a ratio of [A] : [AA] = 0.304 to 0.442 (using total CheA concentration of 5 M).
In vivo, other binding reaction may make this one unimportant.
Using S typhimurium proteins in E coli.
KD = 3 x
10-7 
1x
10-7
Ninfa, 1992
In isolation, this would lead to a ratio of [A] : [AA] = 0.378  2 : 5 (using total CheA concentration of 5 M).
In vivo, other binding reaction may make this one unimportant.


Repellent

Ca2+
Attractant

Ca2+
Ca2+
 Tumbling
R+T
 R-T
KD = 2.5 x 10-6
Tisa & Adler, 1995
Circumstantial evidence demonstrates changes in Ca2+ concentration in response to repellents and
attractants; the way in which this might work is suggested: Ca 2+ could maintain CheY in its phosphorylated
state.
Wu et al, 1996
Though Tar has four methylation sites, there is only one CheR binding site, so only one CheR binds at a
time. Also, covalent modification and dimerisation does not affect Tar to CheR binding (the binding of CheR
to Tar is the same as to TTWWAA and methylated Tar).
In isolation, this would lead to a ratio of [R] : [RT] = 0.516  1 : 2 and a ration of [T] : [RT] = 31 (using total
CheR concentration of 0.235 M and Tar concentration of 5 M).
In vivo, CheR binds to other receptors also.
KD = 2.1 x 10-6
Simms et al, 1991
CheR + TTWWAA  CheR-TTWWAA  CheR + TTmWWAA
KD = 2.1 x 10-6; KM = 4.2 x 10-6; kcat = 0.11 (Simms et al, 1991)
 kass = 5.24 x 104 M-1s-1; kdis = 0.11 s-1
Note that these rates and the dissociation constant could be different when Tar is methylated.
R-T + T
 R-T + Tm
1.90 x
103
Le Moual et al, 1997;
Simms et al, 1987
(From table 2)
Using the rate of methylation for non-aspartate bound receptor complexes from Simms et al, 1987; using
relative methylation rate from Le Moual et al, 1997.
 Rate for asp bound receptor = (1.54 x 10-3) x 0.29 (Divide by [R-T] to get bimolecular rate constant)
Using Tar
2.0 x 103
Wu et al, 1996
It is possible that if CheR is bound to one Tar-containing complex, then it could methylate a Tar in a different
Tar-containing complex. This can be extended to include other receptors, so for instance, a CheR-Tsr
complex could methylate a neighbouring Tar containing complex.
Wu & Weis, 1997
This data is from S typhimurium using Tsr.
Rate of inter-complex methylation is 5.04 x 10-2 methyl (receptor)-1 min-1 compared to intra-complex
methylation of 0.12 methyl (receptor)-1 min-1. Therefore inter-complex methylation represents 30% of total
methylation.
Using a total methylation rate of:
(Total Rate Including CheR) / [CheR] = 6.6 x 103 M-1s-1
where
Total Rate Including CheR = 1.54 x 10-3 (Shapiro et al, 1995)
[CheR] = 0.235 (Simms et al, 1987)
Inter-complex methylation rate = 0.3 x 6.6 x 103 = 2.0 x 103
Intra-complex methylation rate = 0.7 x 6.6 x 103 = 4.6 x 103
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 32
116103993
Reaction
Rate Constant
Source
Comments
R-Ta + Ta  R-Ta + Tma
1.3 x 104
Wu & Weis, 1997
This data is from S typhimurium using Tsr and serine.
In the presence of 2mM serine, rate of inter-complex methylation is 7.8 x 10-2 methyl (receptor)-1 min-1
compared to intra-complex methylation of 0.199 methyl (receptor)-1 min-1. Therefore inter-complex
methylation represents 28% of total methylation.
Using a total methylation rate of:
(Total Rate Including CheR) / [CheR] = 4.5 x 104 M-1s-1
where
Total Rate Including CheR = 1.05 x 10-2 (Simms et al, 1987, Terwilliger et al, 1986)
[CheR] = 0.235 (Simms et al, 1987)
Inter-complex methylation rate = 0.3 x 4.5 x 104 = 1.35 x 104
Intra-complex methylation rate = 0.7 x 4.5 x 104 = 3.15 x 104
2.75 x 104
Le Moual et al, 1997;
Simms et al, 1987
(From table 2)
Using the rate of methylation for non-aspartate bound receptor complexes from Simms et al, 1987; using
relative methylation rate from Le Moual et al, 1997.
 Rate for asp bound receptor = (1.54 x 10-3) x 0.42 (Divide by [R-T] to get bimolecular rate constant)
Using Tar
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 33
Protein Concentrations
Species
Tar
Amount of Protein (cell vol = 1.41 x 10-15 litres)
Particles
Concentration / M
 2,550
 3
 4,200
5
900
 1.06
Source
Comments
Gegner et al, 1992
Total MCP concentration given as 10 M. Tsr is more than half of the total (Hazelbauer & Engstrom,
1981).
Ninfa et al, 1991
No experimental details given.
Hazelbauer & Engstrom,
1981; Hazelbauer et al,
1981; Hazelbauer &
Harayama, 1983
Tar + Tsr level = 2,500 molecules / cell. Tsr is more than half of the total (Hazelbauer & Engstrom,
1981).
This represents 1600 Tsr and 900 Tar (Hazelbauer & Harayama, 1983).
Trg levels are 100-200 (Hazelbauer et al, 1981).
900
 1.06
DeFranco & Koshland, 1981
Also quotes Tsr at 1600 molecules, Trg at 150 molecules, Tap at 150 molecules.
600
 0.71
Clarke & Koshland, 1979
This data is from E coli. Also gives Tsr at 2900 molecules.
 4,250
5
 8,500
10
Gegner et al, 1992
 850
1
Ninfa et al, 1991
No experimental details given.
5
Gegner & Dahlquist, 1991
No experimental details given.
Also gives Tar at 100 molecules and Tsr at 2,000 molecules in S typhimurium.
CheW
 4,200
5,000
CheAL
Matsumura et al, 1990
(P139)
 4,250
5
 8,500
10
Gegner et al, 1992
1
 850
This value is used in BCT (1995), based on: Matsumura et al, 1990; Gegner & Dahlquist, 1991; Ninfa
et al, 1991; Gegner et al, 1992.
Ninfa et al, 1991
No experimental details given.
4,200
 5
Gegner & Dahlquist, 1991
Value is presented as 2.5 M of dimer. No experimental details given.
5,000
 5.88
Matsumura et al, 1990
CheAS concentration is 5.26 M = 4,470 molecules.
 4,250
CheY
 5.88
This value is used in BCT (1995), representing total MCP concentration, based on: Clarke &
Koshland, 1979; Hazelbauer & Engstrom, 1981; Ninfa et al, 1991; Gegner et al, 1992.
15,400
5
 18.1
This value is used in BCT (1995), based on: Matsumura et al, 1990; Gegner & Dahlquist, 1991; Ninfa
et al, 1991; Gegner et al, 1992.
Zhao et al, 1996
 6,750
8
Kuo & Koshland, 1987
 17,000
20
Stock et al, 1985
 8,500
10
Carl J Morton-Firth and Robert B Bourret
This data is from S typhimurium.
This data is from S typhimurium. No experimental details given.
This value is used in BCT (1995), based on: Stock et al, 1985; Kuo & Koshland, 1987.
12 February 2016
Page 34
Species
116103993
Amount of Protein (cell vol = 1.41 x 10-15 litres)
Particles
CheB
1,930
Source
Comments
Simms et al, 1985
This data is from S typhimurium.
Concentration / M
 2.27
Concentration measured as 0.76mg CheB per g cell protein.
Using 1.56 x 10-13 g protein / cell (Neidhardt & Umbarger, 1996) and MR of CheB of 37,000:
Number of molecules = (0.76 x 10-3 ) x (1.56 x 10-13 ) x (6 x 1023 ) / 37,000 = 1,930
CheR
CheZ
1,700
 2
200
 0.235
850
 1
24,100
This value is used in BCT (1995), based on Simms et al, 1985.
Simms et al, 1987
This value is used in BCT (1995).
 28.3
Matsumura et al, 1990
 850
1
Kuo & Koshland, 1987
 17,000
20
12 February 2016
This value is used in BCT (1995), based on: Kuo & Koshland, 1987; Matsumura et al, 1990.
Carl J Morton-Firth and Robert B Bourret
116103993
Page 35
References
Ames, P; Parkinson, J S (1994) J Bacteriol 176:6340-6348
Barak, R; Eisenbach, M (1992a) Biochemistry 31:1821-1826
Barak, R; Eisenbach, M (1992b) J Bacteriol 174:643-645
Barak, R; Giebel, I; Eisenbach, M (1996) Mol Microb 19:139-144
Biemann, H-P; Koshland, D E Jr (1994) Biochemistry 33:629-634
Blat, Y; Eisenbach, M (1994) Biochemistry 33:902-906
Blat, Y; Eisenbach, M (1996a) J Biol Chem 271:1226-1231
Blat, Y; Eisenbach, M (1996b) J Biol Chem 271:1232-1236
Bochner, B R; Ames, B N (1982) J Biol Chem 257:9759-9769
Bogonez, E; Koshland, J E Jr (1985) Proc Natl Acad Sci USA 82:4891-4895
Borczuk, A; Stock, A; Stock, J (1987) J Bacteriol 169:3295-3300
Borkovich, K A; Alex, L A; Simon, M I (1992) Proc Natl Acad Sci USA 89:6756-6760
Borkovich, K A; Kaplan, N; Hess, J F; Simon, M I (1989) Proc Natl Acad Sci USA 86:1208-1212
Borkovich, K A; Simon, M I (1990) Cell 63:1339-48
Bourret, R B (1995) Grant Proposal
Bourret, R B (1996) Personal Communication. 1 May 1996
Bray, D; Bourret, R B (1995) Mol Biol Cell 6:1367-1380
Bray, D; Bourret, R B; Simon, M I (1993) Mol Biol Cell 4:469-482
Bren A; Welch, M; Blat, Y; Eisenbach, M (1996) Proc Natl Acad Sci USA 93:10090-10093
Clarke, S; Koshland, D E Jr (1979) J Biol Chem 254:9695-9702
Clegg, D O; Koshland, D E Jr (1984) Proc Natl Acad Sci USA 81:5056-5060
Conley, M P; Berg, H C; Tawa, P; Stewart, R C; Ellefson, D D; Wolfe, A J (1994) J Bacteriol 176:3870-3877
Conley, M P; Wolfe, A J; Blair, D F; Berg, H C (1989) J Bacteriol 171:5190-5193
Danielson, M A; Biemann, H-P; Koshland, D E Jr; Falke, J J (1994) Biochemistry 33:6100-6109
DeFranco, A L; Koshland, D E Jr (1981) J Bacteriol 147:390-400
Dunten, P; Koshland, D E Jr (1991) J Biol Chem 266:1491-1496
Eisenbach, M (1996) In Press.
Gegner, J A; Dahlquist, F W (1991) Proc Natl Acad Sci USA 88:750-754
Gegner, J A; Graham, D R; Roth, A F; Dahlquist, F W (1992) Cell 70:975-982
Hauri, D C; Ross, J (1995) Biophysical J 68:708-722
Hazelbauer, G L; Engstrom, P (1981) J Bacteriol 145:35-42
Hazelbauer, G L; Engstrom, P; Harayama, S (1981) J Bacteriol 145:43-49
Hazelbauer, G L; Harayama, S (1983) Int Rev Cytology 81:33-70
Hess, J F; Bourret, R B; Oosawa, K; Matsumura, P; Simon, M I (1988 a) Cold Spring Harbor Symp Quant Biol 53:41-48
Hess, J F; Oosawa, K; Kaplan, N; Simon, M I (1988b) Cell 53:79-87
Hess, J F; Oosawa, K; Matsumura, P; Simon, M I (1987) Proc Natl Acad Sci USA 84:7609-7613
Huang, C; Stewart, R C (1993) Biochim Biophys Acta 1202:297-304
Iwama, T; Homma, M; Kawagishi, I (1997) BLAST Conference Poster
Kehry, M R; Doak, T G; Dahlquist, F W (1985) J Bacteriol 163:983-990
Kuo, S C; Koshland, D E, Jr (1987) J Bacteriol 169:1307-1314
Kuo, S C; Koshland, D E, Jr (1989) J Bacteriol 171:6279-6287
Le Moual, H; Quang, T; Koshland, J E Jr (1997) Biochemistry 36:13441-13448
Li, J; Li, G; Weis, R M (1997) Biochemistry 36:11851-11857
Li, J; Swanson, R V; Simon, M I; Weis, R M (1995) Biochemistry 34:14626-14636
Liu, J D; Parkinson, J S (1989) Proc Natl Acad Sci USA 86:8703-8707
Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B (1997) EMBO Journal 16:7231-7240
Lukat, G S; Lee, B H; Mottonen, J M; Stock, A M; Stock, J B (1991) J Biol Chem 266:8348-8354
Lukat, G S; McCleary, W R; Stock, A M; Stock, J B (1992) Proc Natl Acad Sci USA 89:718-722
Carl J Morton-Firth and Robert B Bourret
12 February 2016
Page 36
116103993
Lukat, G S; Stock, A M; Stock, J B (1990) Biochem 29:5436-5442
Lupas, A; Stock, J B (1989) J Biol Chem 264:17337-17342
Marwan, W; Schafer, W; Oesterhelt, D (1990) EMBO J 9:355-342
Matsumura, P; Roman, S; Volz, K; McNally, D (1990) Signalling complexes in bacterial chemotaxis. From Biology of the Chemotactic Response. Ed Armitage, J P; Lackie, J M (CUP)
McNally, D F; Matsumura, P (1991) Proc Natl Acad Sci USA 88:6269-6273
McNamara, B P; Amsler, C; Wolfe, A J (1997) BLAST Conference Poster
McNamara, B P; Wolfe, A J (1997) J Bacteriol 170 In Press.
Milburn, M V; Prive, G G; Milligan, D L; Scott, W G; Yeh, J; Jancarik, J; Koshland, D E Jr; Kim, S-H (1991) Science 254:1342-1347
Milligan, D L; Koshland, J E Jr (1993) J Biol Chem 268:19991-19997
Neidhardt, F C; Umbarger, H E (1996) Chemical composition of Escherichia coli. From Escherichia coli and Salmonella: Cellular and Molecular Biology. Ed Neidhardt, F C (ASM Press, Washington DC)
Ninfa, E G (1992) PhD Thesis. Princeton University, Princeton, NJ
Ninfa, E G; Stock, J (1989) Unpublished results
Ninfa, E G; Stock, J (1991) Unpublished results
Ninfa, E G; Stock, A; Mowbray, S; Stock, J (1991) J Biol Chem 266:9764-9770
Parkinson, J S; Parker, S R (1979) Proc Natl Acad Sci USA 76:2390-2394
Parkinson, J S; Parker, S R; Talbert, P B; Houts, S B (1983) J Bacteriol 155:265-274
Pruss, B M; Wolfe, A J (1994) Mol Microb 12:973-984
Ravid, S; Matsumura, P; Eisenbach, M (1986) Proc Natl Acad Sci USA 83:7157-7161
Russo, A F; Koshland, D E Jr (1983) Science 220:1016-1020
Sanatinia, H; Kofoid, E C; Morrison, T B; Parkinson, J S (1995) J Bacteriol 177:2713-2720
Sanna, M G; Swanson, R V; Bourret, R B; Simon, M I (1995) Mol Microb 15:1069-1079
Schuster, M; Abouhamad, W N; Silversmith, R E; Bourret, R B (1997) Mol Microb In Press
Schuster, S C; Swanson, R V; Alex, L A; Bourret, R B; Simon, M I (1993) Nature 365:343-347
Shapiro, M J; Koshland, D E Jr (1994) J Biol Chem 269:11054-11059
Shapiro, M J; Panomitros, D; Koshland, D E Jr (1995) J Biol Chem 270:751-755
Shukla, D; Matsumura, P (1995) J Biol Chem 270:24414-24419
Silversmith, R (1996) Personal Communication. 25 October 1996
Silversmith, R; Appleby, J L; Bourret, R B (1997) Biochem In Press.
Simms, S A; Keane, M G; Stock, J (1985) J Biol Chem 260:10161-10168
Simms, S A; Stock, A M; Stock, J B (1987) J Biol Chem 262:8537-8543
Simms, S A; Subbaramaiah, K (1991) J Biol Chem 266:12741-12746
Smith, J M; Roswell, E H; Shioi, J; Taylor, B L (1988) J Bacteriol 170:2698-2704
Springer, M S; Zanolari, B (1984) Proc Natl Acad Sci USA 81:5061-5065
Stewart, R C (1993a) J Biol Chem 266:1921-1930
Stewart, R C (1993b) Personal Communication. 2 February 1993
Stewart, R C (1993c) Personal Communication. 3 February 1993
Stewart, R C (1997a) BLAST Conference Presentation
Stewart, R C (1997b) Biochem In Press.
Stewart, R C; Dahlquist, F W (1987) Chem Rev 87:997-1025
Stewart, R C; Roth, A F; Dahlquist, F W (1990) J Bacteriol 172:3388-3399
Stock, A; Koshland, D E Jr; Stock, J (1985) Proc Natl Acad Sci USA 82:7989-7993
Stock, J B; Koshland, D E Jr (1981) J Biol Chem 256: 10826-10833
Stock, J B; Lukat, G S; Stock, A M (1991) Annu Rev Biophys Biophys Chem 20:109-136
Stock, J B; Lukat, G S; Stock, A M (1991) Annu Rev Biophys Biophys Chem 20:109-136
Stock, J B; Ninfa, A J; Stock, A M (1989) Micro Rev 53:450-490
Stock, J B; Surette, M G (1996) Chemotaxis. From Escherichia coli and Salmonella Cellular and Molecular Biology. Ed Neidhardt, F C (ASM Press, Washington, DC). 1103-1129
Surette, M G; Levit, M; Liu, Y; Lukat, G; Ninfa, E G; Ninfa, A; Stock, J B (1996) J Biol Chem 271:939:945
Surette, M G; Stock, J B (1996) J Biol Chem 271:17966-17973
Swanson, R V; Bourret, R B; Simon, M I (1993) Mol Microb 8:435-441
Swanson, R V; Schuster, S C; Simon, M I (1993) Biochem 32:7623-7629
Tawa, P; Stewart, R C (1994) Biochemistry 33:7917-7924
12 February 2016
Carl J Morton-Firth and Robert B Bourret
116103993
Page 37
Terwilliger, T C; Wang, J Y; Koshland, D E (1986) J Biol Chem 261:10814-10820
Timasheff, S N (1993) Annu Rev Biophy Biomol Struct 22:67-97
Tisa, L S; Adler, J (1995) Proc Natl Acad Sci USA 92:10777-10781
Wang, E A; Koshland, D E, Jr (1980) Proc Natl Acad Sci USA 77:7157-7161
Wang, H; Matsumura, P (1996) Mol Microb 19:695-703
Welch, M; Oosawa, K; Aizawa, S I; Eisenbach, M (1993) Proc Natl Acad Sci USA 90:8787-8791
Welch, M; Oosawa, K; Aizawa, S I; Eisenbach, M (1994) Biochem 33:10470-10476
Wolfe, A J; McNamara, B P; Stewart, R C (1994) J Bacteriol 176:4483-4491
Wolfe, A J; Stewart, R C (1993) Proc Natl Acad Sci USA 90:1518-1522
Wu, J; Li, J; Li, G; Long, D G; Weis, R M (1996) Biochem 35:4984-4993
Wu, J; Weis, R M (1997) BLAST Conference Poster
Wylie, D; Stock, A; Wong, C Y; Stock, J (1988) Biochem Biophys Res Comm 151:891-896
Yamaguchi, S; Aizawa, S; Kihara, M; Isomwa, M; Jones, C J; Macnab, R M (1986) J Bacteriol 168:1172-1179
Yeh, J I; Biemann, H P; Pandit, J; Koshland, J E Jr; Kim, S H (1993) J Biol Chem 268:9787-9792
Yonekawa, H; Hayashi, H (1986) FEBS Lett 198:21-24
Zhao, R; Amsler, C D; Matsumura, P; Khan, S (1996) J Bacteriol 178:258-265
Zimmerman, S B; Minton, A P (1993) Annu Rev Biophy Biomol Struct 22:27-65
Carl J Morton-Firth and Robert B Bourret
12 February 2016