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

150

200

250

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

500

1000

1500

2000

2500

0 100 200 300 400 500

Time (sec)

RU

0

500

1000

1500

2000

2500

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