pre-steady-state kinetics vs steady-state kinetics 1.the order of binding of substrates and release...
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Pre-steady-state kinetics vs steady-state kinetics 1. The order of binding of substrates and release of product serves to define the reactants present at the active site during catalysis: it does not establishthe kinetically preferred order of substrate addition and product release orallow conclusions pertaining to the events occurring between substrate bindingand product release.2. The value of kcat sets a lower limit on each of the first-order rate constantsgoverning the conversion of substrate to product following the initial collisionof substrate with enzyme. These include conformational changes in the enzyme-Substrate complex, chemical reactions (including the formation and breakdownof intermediates), and conformational changes that limit the rate of product release.3. The value of kcat/KM defines the apparent second-order rate constant for substrate binding and sets a lower limit on the second-order rate constant forsubstrate binding. The term kcat/KM is less than the true rate constant by a factor defined by the kinetic partitioning of the E-S to dissociate or go forward in the reaction.
The goal of pre-steady-state kinetics to to establish the complete kinetic pathwayIncluding substrate binding, chemical reaction (substrate through intermediates to product), and product release.
E+ S ES EX EP E + Pk1
k2 k3 k4
k-1 k-2 k-3 k-4
Fast kinetics•Product release step is slow so the steady-state rate = product release rate
•To measure the rate of chemical step where the product release is much slower, a single-turnover condition needs to be employed.
•Under single-turnover condition where [E] >[S], product release needs not to be considered.
•Under multiple-turnover condition where [S] = 4 x [E], a burst kinetics (a fast phase followed by a steady-state phase of product formation) can be observed for a reaction with slower post-chemical step.
•A special tool Quench-Flow, needs to be used for single-turnover experiment in msec time scale.
•A Stopped-Flow instrument allows the measurements of
ligand interaction and chemical steps.
Rapid-Quench fast kinetics instrumentMeasure the real rate of chemical step (single turnover, [E]>[S])
Measure the product formation burst (multiple turnover, [S] = 4x[E])
UPPs (undeca-prenyl pyrophosphate synthase) reaction
UPPs catalyzes sequential addition of eight IPP to an FPP molecule, forming an undeca-prenyl pyrophosphate with 55 carbons and newlyformed cis double bonds.
UPPs synthesizes lipid carrier for bacterial cell wall assembly
Dolichyl pyrophosphate synthase catalyzes the lipid carrier for Glycoprotein syntehsis
lipid I
Steady-state kinetics
Enzyme + FPP + [14C]IPP
Incubate
differentperiods
butanol [14C]products
[14C]IPP
Counting radiolabel in butanol vs. buffer to determine rate constant kcat = 0.013 s-1 (without Triton)kcat = 2.5 s-1 (with Triton)
[14C]IPP
[14C]products
counting
counting
E EE
Substrate
binding
reaction
Product release
190-fold increase
Reaction or product release is rate limiting?
Rapid-Quench fast kinetics instrumentMeasure the real rate of chemical step under single turnover, ([E]>[S])
E EEE
Stop the reaction in msec time scale
Rate is not limitedby product release.
Rapid-Quench fast kinetics to measure the rate constants of IPP condensation
0
2
4
6
8
10
0 2 4 6 8 10Time (sec)
Con
cen
trat
ion
(uM
)
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6
Con
cent
rati
on (
uM)
Time (sec)
Y axis represents the sum of [14C]IPP incorporated
10 M UPPs, 1 M FPP, 50 M [14C]IPPSingle turnover experiments
Time courses of C20 (●), C25 (○), C30 (■), C35 (□), C40 ( ), C45 (◊), C50 (▲), and C55 ( ).◆ △
Pan et al., (2000) Biochemistry 10936-10942
evaporateAcidic phosphatase
Buffer (pH 4.8)
hexane
Mobile phase H2O:acetone = 1:19
Evaporate to
Small volume
Product Analysis using TLC
PPi OH
OH
Reverse phaseTLC
butanol
The rate constants for IPP condensation determined from single-turnover
IPP
IPP
IPP
IPP
IPP
IPP
IPP
IPP
E + FPP E-FPPfast
fastE-FPP-IPP E-C20
E-C20-IPPE-C25E-C25-IPPE-C30
E-C30-IPP E-C35 E-C35-IPP E-C40
E-C40-IPPE-C45E-C45-IPPE-C50
E-C50-IPP E-C55 E + C55
2.5 s-1
2 s-13.5 s-1
2.5 s-13 s-1
3.5 s-13.5 s-1
3 s-1 fast (with triton) but slow (w/o)
fast
Steady-state kcat is 2.5 s-1 in the presence of 0.1% Triton, which is consistent with IPP condensation rate; and is 190-fold larger than the rate constant (0.013 s-1) in the absence of Triton.
No Triton
E E
Multiple turnovers ([S] = 2x[E]) measured by Rapid-Quench
E E
0.75 M enzyme, 6 M FPP and 50 M [14C]IPP without Triton
0
2
4
6
8
10
0 20 40 60 80 100 120 140 160Time (sec)
Con
cent
ratio
n (u
M)
-0.1
0
0.1
0.2
0.3
0.4
0.5
0 20 40 60 80 100 120 140 160Time (sec)
Con
cent
ratio
n (u
M)
The data indicate formation of C55 (△), C60 (●), C
65 (■), C70 (◆) and C75 (▲)
IPP
IPP
IPP (without triton)
E-C55-IPP
E-C60
E-C60-IPP
0.4 s-1
E-C65
E-C65-IPP
0.4 s-1
E-C70
0.001 s-1
E* + C60
E* + C65
E + C70 + C75
E0.001 s-1
E0.001 s-1
E-C550.4 s-1
E* + C55E0.001 s-1
0.5 s-1
0.1 s-1
0.02 s-1
UPPs kinetic scheme after product is formed (W/O Triton)So the kcat = 0.013 s-1
In the cells, membrane lipidmay play a role of Triton to control the C55 chain length
Fluorescent probe for ligand interaction and inhibitor binding using stopped-flow
OPP
PPO-OP
O
O-
O
P
O
O-
O
A B
Flow Cell
Light
Stop SyringeFluorescence Signal
Absorbance Signal
E
E-P excess probe
Synthesis of fluorescent substrate analogue
OH O O O O
OH
O O
Br
OO
OO
CF3
O
OOH
OO
CF3
OBr
OO
CF3
OO
OO
CF3
PO
OP
O-
O
O-O-
1 2 3
4
(a) DHP, PPTs, CH2Cl2, 90%; (b) SeO2, t-BuOOH, salicylic acid, CH2Cl2, 32%; (c) NBS, Me2S, CH2Cl2, 92%; (d) K2CO3, DMF,7-hydroxy-8-methyl-4-trifluoromethylcoumarin, 94%; (e) PPTs, EtOH, 92%; (f) CBr4, PPh3, CH2Cl2, 80%; (g) (n-Bu4N)3HP2O7, CH3CN, 46%.
a b
c d
e f
g
3 NH4+
5
6 7
8
7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4-trifluoromethyl-chromen-2-one geranyl pyrophosphate (TFMC-GPP)
Chen et al., (2002) J. Am. Chem. Soc. 124, 15217-15224
OOPP
OO OPP
CF3
400 450 500 550 6000
200
400
600
800
1000
1200
F
luor
esce
nce
Inte
nsity
(a.
u.)
Wavelength (nm)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
200
300
400
500
600
700
Flu
ores
cenc
e In
tens
ity (
a.u.
)
Concentration ()
Interaction of the fluorescent probe with UPPs
(A) Fluorescence is quenched by UPPs and recovered by replacement with FPP(B) Probe binds to UPPs with 1:1 stoichiometry
(A) (B)
(C) (D)
(C ) Probe binds to UPPs with a kon = 75 M-1 s-1
(D) Probe releases from UPPs (chased by FPP) with a koff = 31 s-1
Kinetic scheme for UPPs reaction
FPP is released at 30 s-1 UPP is released at 0.5 s-1
The rate constants ~2.5 s-1 for each IPP condensation were derived from Rapid-Quench experiments.
IPPE + FPP E-FPP
fast
30 s-1E-FPP-IPP E-C20
E-C25 E-C30 E-C35 E-C40
E-C45 E-C50 E-C55 E + C55
2.5 s-1 2 s-1
3.5 s-1 2.5 s-1 3 s-1 3.5 s-1
3 s-13.5 s-1 0.5 s-1
2 M-1 s-1
Binding order and conformational changeusing fluorescence detection
3-D structure of E. coli UPPs
Two conformers were found: one (closed form) with PEG bound and the other (open form) has empty active site, and the 3-B loop is invisible
Ko et al., (2001) J. Biol. Chem. 47474-47482
300 320 340 360 380 400 420 4400
500
1000
1500
2000
2500
3000
3500F
luo
resc
en
ce I
nte
nsi
ty (
a.u
.)
Wavelength (nm)
300 320 340 360 380 400 420 4400
500
1000
1500
2000
2500
3000
3500
Flu
ore
sce
nce
In
ten
sity
(a
.u.)
Wavelength (nm)
300 320 340 360 380 400 420 4400
500
1000
1500
2000
2500
3000
3500
Flu
ore
sce
nce
In
ten
sity
(a
.u.)
Wavelength (nm)
FPP binding induces conformational change on 3 helix
wild-type W31F has less quench
W91F has almost no quench Chen et al., (2002) J. Biol. Chem. 7369-7376
FPP bound crystal structure
Chang, S. Y. (2004) Protein Sci. 971-978
FPP
In UPPs reaction, FPP binding does not require Mg2+
and IPP binding needs Mg2+ (also FPP binds first)
FPP (or FsPP) quenches the UPPs Intrinsic fluorescence even in the absence of Mg2+
+Mg2+
Mg2+ is required fro IPP binding
No Mg2+
UPPs conformational change during catalysis
Flexible loop
32
1
O P O P O-
-O-O
OO
-O
PO
O-
OP
O
O-
OFlexible loop
32
1
closed-form open-form
Chain elongation
W75W31
bindingrelease
L137
W91
-O
P
O
O-
OP
O
O-
O
Chen et al., (2002) J. Biol. Chem. 7369-7376
E-FPP -> <- IPP (Mg2+)
10 sec 200 msec 200 msec
E-FsPP -> <- IPP (Mg2+)IPP kon = 2 uM-1s-1
Reach the top at 2 sec when the product is formed
E + FPP is too fastto be observed.
Mutations on some residues of the flexible loop (71-83) or change the loop length affect UPPs activity
UPPs kcat (s-1) Km (FPP) (M) Km (IPP) (M) relkcat
a
wild type 2.5 0.1 0.4 ¡Ó 0.1 4.1 ¡Ó 0.3
133 ¡Ó 14
16.2 ¡Ó 2.2
1.0 ¡Ó 0.2
0.4 ¡Ó 0.1
8 ¡Ó 0.6
1.6 ¡Ó 0.3 15.7 ¡Ó 2.5
1
0.04
0.1
0.01
0.30 0.01
S71A
E73A
N74A
akcat relative to that of wild type
¡Ó
¡Ó
E81A 0.4 ¡Ó 0.1 0.4 ¡Ó 0.1 88 ¡Ó 10 0.2
0.4 ¡Ó 0.1(2.20 ¡Ó 0.03) x 10-2
R77A (1.4 ¡Ó 0.1) x 10-4 5 x 10-5
0.11 ¡Ó 0.01
W75A 1.1 ¡Ó 0.1 3.2 ¡Ó 0.3 46 ¡Ó 4 0.5
S83(Ala)5 1.3 x 10-4 0.17 ¡Ó 0.03 7.8 ¡Ó 2.3 10-4
S83(Ala)1 9.0 x 10-4 0.43 ¡Ó 0.2 34 ¡Ó 5 10-4
£GV82S83 2.2 x 10-4 1.8 ¡Ó 0.16 > 3600 10-4
£GS83 7.6 x 10-5 1 ¡Ó 0.15 > 3200 10-4
£GS72 2.8 x 10-5 0.5 ¡Ó 0.13 11.7 ¡Ó 2.6 10-4
Ko et al., (2001) J. Biol. Chem. 277, 47474-47482Chang et al., (2003) Biochemistry 42, 14452-9
Flexible loop
32
1
O P O P O--O
-O
OO
-O
PO
O-
OP
O
O-
O
Flexible loop
32
1
w.t. closed-formactive
w.t. open-forminactive
release
W91
Elongated loop
32
1
insertion mutantopen-forminactive
32
1
deletion mutant open-forminactive
B B
B B
Shorten loop
UPP
(A)
(B) (C)
FPP FPP
Both insertion and deletion mutants adopt open form
S83(Ala)5
V82S83
(A) Wild type
(B) S83(Ala)5 (C) V82S83
UPPs in complex with sulfates, and two Triton
Chang et al., (2003) J. Biol. Chem. 278, 29298-29307Two Tritons
C55 (8 cis)
Open formArg30
Arg39Arg194
Arg200
Co-crystal structures of UPPs complexed with FsPP and Mg2+IPP(closed form)
Guo et al., (2005) J. Biol. Chem
Conformational changes
In the active site,Mg2+ is bound with PPi of FPP
Two positions of Mg2+ in several crystal structures
S1 and S2 are SO4 ions which solved from previous studies.Yellow: UPPs bound with FPP only
D26A-IPPMg-IPP-FsPP
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 50 100 150
IPP
inco
rpo
ratio
n ra
te (
μM
/min
)
[MgCl2] (mM )
-5
0
5
10
15
20
25
30
35
-0.5 0 0.5 1 1.5 2 2.5 3
1/ [ Mg-IPP] (μ M -1)
1/V
(min
/μM
) Mg 0.05mM
Mg 1mM
Mg 3mM
Mg 50mM
[Mg2+] dependence of enzyme activity
Ki = 1 mM
300 350 400 450 5000
50
100
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200
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300
Rel
ativ
e F
luor
esce
nce
Inte
nsity
(a.
u.)
Emission Wavelength (nm)
300 350 400 450 5000
50
100
150
200
250
300
Re
lativ
e F
luo
resc
en
ce In
ten
sity
(a
.u.)
Emission wavelength (nm)
+Mg2+ (1 mM), IPP binding
w/o or w 50 mM Mg2+, no IPP binding
Proposed reaction mechanism of UPPs reactionMg2+-IPP in
D26 Mg2+-D26-FPP
Condensation reaction occurs
Mg2+-PPi out
How to measure protein-ligand interaction?
1. Measure kon, koff and Kd = koff / kon using the instruments such as stopped-flow and BIAcore etc.
2. Measure the Kd using titration (fluorescence titration, isothermal titration calorimetry etc).
3. ELISA assays to measure the MIC (minimum inhibitory concentration).
4. Measure the protein oligomerization status and the Kd using AUC (analytic ultra-centrifuge).
5. Km from Michaelis-Menten kinetics.’6. Equilibrium gel filtration.7. Equilibrium dialysis.
Structural Requirement and Ligand Specificity of Tachypleus Plasma Lectins (TPL) for Molecular Recognition in Host Defense
Horshoe crab, an arthropod dependent on innate immunity.
TPL-1 and TPL-2 were previouslyIsolated from plasma of horseshoe crab
LPS detection kit
Defense mechanism in horseshoe crab
Lectins : biosensors,
immobilize and help killing invaders
Previous finding, by Liu et al.
LPS-4B resin
Horseshoe crab plasma
CL-4B resin
Tachypleus plasma lectin
(TPL-1)
TPL-2
Chiou et al., J. Biol. Chem (2000)
Sequence alignment for TPL-1
N glycosylation site
Chen et al., J. Biol. Chem. (2001)
Sequence alignment for TPL-2
N glycosylation site
SDS-PAGE of purified wild-type and mutant TPLs
reducing Non-reducing
Lane 1: MW standardsLane 2: TPL-1Lane 3: TPL-1-N74DLane 4: TPL-2Lane 5: TPL-2-N3DLane 6: TPL-2-C4SLane 7: TPL-2-C6SLane 8: TPL-2-C64S
Kuo et al., Biochem J. (2006)
ELISA Procedure
E. coli Bos-12 Serial diluted TPL-1 or -2
Add 1st TPL-1 or -2 Ab and then HRP-linked 2nd Ab
Incubate and wash
Add HRP substrate
Detect OD 450
TPL-1-N74D is inactive and TPL-2-N3D is active
: TPL-1
: N74D TPL-1
: TPL-2
: N3D TPL-2
Kuo et al., Biochem J (2006)
ligandsMIC to TPL-1 (mM)
MIC to TPL-2 (mM)
Glucose >50 >50
Galactose >50 >50
Mannose >50 >50
Fucose >50 >50
Maltose >50 >50
Lactose >50 >50
GlcNAc 8.4 >50
ManNAc 12.3 >50
GalNAc 18.5 >50
NANA 32 >50
LacNAc 14.3 >50
Acetic acid >50 >50
L-glutamine >50 >50
N-acetyl-glutamine 50 >50
N,N-Dimethyl-acetamide >50 >50
N,N-Diacetyl-chitobiose 9.3 >50
N,N,N,N,N-pentaacetyl-chitopentaose
7.2 50
Peptidoglycan monomer 0.078 ND
peptidoglycan
0
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2500
0 100 200 300 400 500
Time (sec)
RU
0
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1500
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3000
0 100 200 300 400 500
Time (sec)R
U
BIAcore Measurements
TPL-1 bound peptidoglycan unit (digested by LytG) with KD = 8 x 10-8 M (left panel); and bound muramyl-dipeptide with KD = 2.9 x 10-7 M (right panel). TPL-2 bound LPS with KD = 6.3 x 10-8 M (not shown).
ligands MIC to TPL-1 (mg/ml)
MIC to TPL-2 (mg/ml)
Lipid A E. coli F583 (Rd) >1 >1
Ra mutant LPS E. coli EH100
ND >1
Delipidized LPS O128:B8 ND 0.16
LPS E. coli K-235 ND 0.3
LPS E.coli 026:B6 >1 0.22
LPS E. coli 055:B4 >1 0.15
LPS E. coli 0111:B4 >1 0.28
Peptidoglycan 0.12 >1
Mannan >1 >1
Lipotechioic acid >1 >1
Peptidoglycan is target for TPL-1 and LPS is recognized by TPL-2
LPS
MIC for TPL-1 and TPL-2 in presence of 1mM DTT
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6
A45
0
TPL-1 conc. (uM)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 1 2 3 4 5 6
A45
0
TPL-2 conc (uM)
● : without DTT■ : with 1mM DTT
● : without DTT■ : with 1mM DTT
●
●
●
●
●
●
●
●●
■
■
■
■
■
■ ■ ■ ■ ■
LPS induces aggregation of TPL-2C4S is monomer and C6S is dimer
Structural model for functional TPL-2
Applications of TPLs in detecting bacteria and removing endotoxin
100 bacteria/ml can inhibit binding of TPLs to bacteria
TPL-2 can inhibit the growth of gram(-) E. coli TPL-2 can be used to remove endotoxin