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Biomolecules: amino acids, peptides, proteins, nucleic acids,
Table: Structures and abbreviations of the standard amino acids of proteins, and
the pK values of their ionization groups
_________________________________________________________________
Name
Structure
pK1 (COOH)
pK2 (NH3+)
pKR (side cahin)
_________________________________________________________________
with nonpolar side chains
Glycine (Gly)
2.35
9.78
Alanine (Ala)
2.35
9.87
Valine (Val)
2.29
9.74
Leucine (Leu)
2.33
9.74
Isoleucine (Ile)
2.32
9.76
Methionine (Met)
2.13
9.28
Proline (Pro)
1.95
10.64
Phenylalanine (Phe)
2.20
9.31
Tryptophan (Trp)
2.46
9.41
With uncharged polar side chains
Serine (Ser)
2.19
Threonine (Thr)
2.09
Asparagine (Asn)
2.14
Glutamine (Gln)
2.17
Tyrosine (Tyr)
2.20
Cysteine (Cys)
1.92
9.21
9.10
8.72
9.13
9.21
10.70
10.46 (phenol)
8.37 (sulfhydryl)
__________________________________________________________
With charged polar side chains
Lysine (Lys)
2.16
9.06
10.54 (e-NH3+)
Arginine (Arg)
1.82
8.99
12.48 (guanidino)
Histidine (His)
1.80
9.33
6.04 (imidazole)
Aspartic acid (Asp)
1.99
9.90
3.90 (b-COOH)
Glutamic acid (Glu)
2.10
9.47
4.07 (g-COOH)
__________________________________________________________________
HA
H+ + A- (equilibrium constant is K = [H+][A-]/[HA], pK = -log K
[H+] = K [HA]/[A-], so -log [H+] = -log K + log [A-]/[HA], so pH = pK + log [A-]/[HA]
When pH = pK, [A-] = [HA]
For a buffer, in the titration curves, when [HA] = [A-], the pH of the solution
is relatively insensitive to the addition of strong base or strong acid.
Proteins
Biosynthesis: DNA transcription mRNA translation proteins
Primary structures: sequences
Secondary structures: a-helices, b-strands etc.
Tertiary structures: 3-D structures
Quaternary structures: compositions, such as dimer, tetreamer etc.
Stabilizing interactions in macromolecules
1. Covalent bonds
2. Electrostatic interactions
3. Dipole-dipole interactions
4. Van der Waals interactions
5. Hydrogen bonds
6. Hydrophobic interactions
Table: Average Dissociation Energies of Chemical Bonds
______________________________________________
Bond type
Bond Energy (kJ/mol)
C-H
408
C-C
342
C=C
602
C=N
606
C=O
732
O-H
458
Enzyme catalysis
1.
2.
3.
4.
5.
6.
Acid-base catalysis
Covalent catalysis
Metal ion catalysis
Electrostatic catalysis
Proximity and orientation effects
Preferred binding of the transition state complex
1. Acid-base catalysis
CH2OH
H A
O
O
H
H
H
OH
C
O H
CH2OH
H
OH
:B-
H
H
OH
H
a-D-Glucose
H
O
H-B
O
OH
C
H
H :B-
a-Pyridone involves
the reaction
OH
N
O
N
CH2OH
H :A
HC
H
H A
OH
b-D-Glucose
-
O
H
OH
OH
OH
H
O
O
H
OH
H
OH
H
OH
H
OH
linear form
O
H
O
O
C
H :B-
H
H
O
O
C
H
v=k[a-pyridone][tetramethyl-a-D-glucose]
The bovine pancreatic RNase A-catalyzed hydrolysis of RNA
O
P O CH2 O Base
O
H
H
H
H
NH
H2O
N
His 12
O OH
O P O CH2
O
Base
O
HO CH2
H
H
O
H
Base
H
N+
H
H
OH
O
H
O P O
N
OH
O
H
O
O P O
His 119
O
O
P O CH2 O Base
O
H
H
H
H
NH
H+N
O O
His 12
P
O
HO
H
N H O
N
H
His 119
O
P O CH2 O Base
O
H
H
H
H
NH
N
O OH
His 12
O P OH
H
O
N+
N
H
His 119
2. Covalent catalysis
H
+
N
H H
N C OH
H
O
H
H-A
:B
Lys
HC
2-
O3PO
+
N
N+
O
CH2 C
CO2
+ OH-
H+
O-
O
RNH2
OH-
OHCO2
H
H2C
CH3
Acetone
Enolate
RNH2
R
O
H2C
CH2
H2C
-
acetoacetate
H
OCH3
N C
Schiff base (w PLP)
O
H2C
H
N+
H
O
CH2 C
O-
Schiff base (imine)
R
H
+
CH2
H
N
+
H
N
H2C
R
H2C
CH3
3. Metal ion catalysis
1. Metalloenzymes: containing tightly bound metal ions, most commonly transition
metal ions such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or Co3+
2. Metal-activated enzymes: loosely bind metal ions from solution, usually the alkaline
earth metal ions Na+, K+, Mg2+, or Ca2+
Three major roles:
1. By binding to substrates so as to orient them properly for reaction
2. By mediating oxidation-reduction reactions through reversible changes in the
metal ion’s oxidation state.
3. By electrostatically stabilizing or shielding negative charges
Mn+
O CH3
OC
O
Mn+
C C
CH3
C
-
O
O
Im
Im
O-
O-
O
2+
CH3
-
C
CH3
Zn
O
C
Im
H
O
O
O
Im
H O C
O-
Im
O
Mg2+
O
O-
Adenine Ribose O P O P O P OO
O
CH3
O
Zn2+
Im
-
CH3
C CH
O
-
O
O
+ Mn+
4. Electrostatic catalysis
The pK’s of amino acid side chains in proteins may vary by several units
from their nominal values
5. Proximity and orientation effects
a. Proximity alone contributes relatively little to catalysis
b. Properly orienting reactants and arresting their relative motions can
result in large catalytic rate enhancement
O
H3C
O
NO2
k1
N
k2 = 24 x k1[imidazole]
NH
O
H2
C
O
NO2
k2
N
NH
R
R'
R''
Y-
R'
R
R'
R''
R
R''
Y-
Y
6. Preferred binding of the transition state complex
Transition state analogues are competitive inhibitors
proline racemase
-
COO
C
N
H
H
C
H
+
+
H
N
H
H
planar TS
COO-
D-proline
L-proline
C- COON
H
COON
H
N+
H
pyrrole-2-carboxylate
competitive inhibitors
COO-
D-1-pyrroline-2-carboxylate
Lysozyme
A. Enzyme structure: E + (NAG)3 poor substrate
(NAG)6 is a good substrate, 2-fold smaller kcat than (NAG-NAM)3
Modeling suggested that the fourth NAG needs to be distorted to change to half-chair form
Asp52 and Glu35 are close to the cut. For non-enzymatic reaction, oxonium ion can be formed.
OR'
H C OR'
OR'
H C O R"
R H
+
+H
R
R"OH
acetal
R'
O+
C
H R
R'
OR'
H C OH
O
C+
H R
Hemiacetal
R
oxonium ion (resonance-stabilized)
When the reaction was run in 18O water,
18O
O
was incorporated.
NAM
RO
CH2OH
C
O+
O
H3C
N
H
O
-
OH
H O
C Glu35
O
NAG HO
O
H3C
C Asp52
-
O
C
O
O
N
H
O
Possible covalent catalysis (need proof)
CH2OH
H
O
O
O O C CH2
H
OR
H
H
NHCOCH3
Asp52
covalent catalysis
H
Intermediate can be trapped by speeding up its formation and slowing down its decomposition.
CH2OH
CH2OH
O
H
H
OH OH
H
H
O
O
H
H
OH
NHCOCH3
H
H
H
F
H
(good leaving group)
-
O
O C CH2-Asp52
F
(stabilize the negative charge)
MASS and crystal structure showed unambiguously the intermediate formation
Serine protease
Burst kinetics
assay
chymotrypsin
O
H3C
O
NO2
p-nitrophenyl acetate
fast
rapid
O
H3C
Enzyme
acy-enzyme intermediate
+
-
O
NO2
450 nm
slow
O
H3C
O-
Burst kinetics: A rapid release of p-nitrophenylacetate followed by a slow release of acetate
Asp-His-Ser catalytic triad and oxyanion hole to facilitate tetrahedral intermediate
The tetrahedral intermediate is mimicked in a complex of Trypsin with Trypsin inhibitor
1013
M-1
Trypsin-BPTI (bovine pancreatic trypsin inhibitor)
The side-chain oxygen of Ser95 is in closer than van der Waals contact
with the pyramidally distorted carbonyl carbon of BPTI’s scissile peptide
KA =
Ser195
H
Ala 16I
O
Ca
O
C N
Ca
Lys 15I
H
Asp102
His57
H2C C O
O-
Asp102
Ser195
H2C
H2C C O
O-
1
H N
CH2
N
His57
H N
H O
N
C
H O
R
R'
O
H
CH2
N+
R
R'
Ser195
H2C
N
C
O-
H
Tetrahedral intermediate
Asp102
H2C C
His57
O
O-
Asp102
Ser195
H2C
H2O
H N
CH2
N
H2C C O
O-
His57
H N
CH2
N
H O
R'NH2
R
O
Ser195
H2C
C
R
N
O
H
H O
R'
C
O
H
Acyl-enzyme intermediate
Asp102
H2C C O
O-
Asp102
His57
Ser195
H2C
H N
CH2
+
N
H2C C O
O-
His57
Ser195
H2C
H N
CH2
N
H O
H O
R
O
H
R
C
O-
O
H
C
O
Thermodynamics and Kinetics in Biology
Thermodynamics (Greek: therme, heat + dynamis, power)
A. The First Law of Thermodynamics: Energy Is Conserved
A system is defined as the part of universe that is of interest, such as
a reaction vessel or an organism; the rest of the universal is known
as the surroundings.
H (enthalpy) = U (energy) + PV
At constant pressure, DH = DU + PDV = qp – w + PDV = qp – PDV +
PDV = qp (heat change)
B. The Second Law of Thermodynamics: Entropy Tends to Increase
The degree of randomness of a system is indicated by its entropy (S).
S = kB ln W (KB = Boltzmann constant; W = the number of energetically
equivalent ways)
DS universe > 0
C. Free Energy
DS > qp/T = DH/T, thus DH – TDS < 0
In 1878, by J. Willard Gibbs, he defined the Gibbs free energy, G = H – TS
Spontaneous processes at constant temperature and pressure have
DG = DH – TDS < 0 (exergonic)
When DG = DH – TDS > 0 (endergonic); DG = DH – TDS = 0 (equilibrium)
Table: Variation of reaction spontaneity (sign of DG with signs of DH and DS
_____________________________________________________________
DH
DS
DG = DH-TDS
_____________________________________________________________
+ The reaction is both enthalpically favored (exothermic) and entropically favored.
The reaction is enthalpically favored (exothermic) and entropically opposed.
+
+ The reaction is both enthalpically opposed (endothermic) and entropically favored.
+
The reaction is both enthalpically and entropically opposed.
_____________________________________________________________
D. Chemical Equilibrium and the Standard State
For a reaction aA + bB
cC + dD
DG = cGC + dGD –aGA –bGB
DGo = cGCo + dGDo –aGAo –bGBo (reactants and products are in their standard states)
DG = DGo + RT ln ([C]c[D]d/[A]a[B]b)
At equilibrium DG = 0, so DGo = -RT ln Keq
Keq = exp (-DGo/RT)
Keq depends on Temperature
ln Keq = -DHo/R(1/T) + DSo/R, the plot of ln Keq versus 1/T is known as a
van’t Hoff plot.
Standard State:
1. Temperature at 25 oC, at neutral pH =7, pressure at 1 atm
2. Consider [H2O] = 1
Elementary Reactions
A I 1 I2
P (I: intermediates)
P (A: reactant, P: product)
k
aA + bB + …….+zZ P Rate = k [A]a[B]b…..[Z]z order: a+b+…+z
A
A
P
A+B
v = d[A]/d[t] = k[A] (first-order reaction, k = s-1)
P
v = d[A]/d[t] = d[B]/d[t] = k{A][B] (second-order reaction, k= M-1s-1)
For first-order Rx: d[A]/[A] = -k d[t]
For second-order Rx A+A
P
ln[A] = ln[A]o - kt
d[A]/[A]2 = k d[t]
[A] = [A]oe-kt
t1/2 = ln2/k
1/[A] = 1/[A]o + kt
Transition state theory of enzyme Catalysis
Activation energy profile of a reaction.
(a) Activation energy (DGo╪)
, free energy change (DGo)
(b) A comparison of activation energy profiles
for catalyzed and uncatalyzed reactions.
For a reaction A + B  P
Rate = -DA/Dt = -DB/Dt = DP/Dt
= k+[A][B] – k-[P]
k = (kT/h) exp (-DG ╪ /RT)
k=Boltzmann constant, h=Planck constant
R: gas constant)
DGo = -RT lnKeq (Keq = [P]/[A][B])
Keq = k+ / k- (forward reaction rate constant
/ reverse reaction rate constant )
Steady-state Enzyme Kinetics (simplified scheme)
E+S
k1
ES
k2
E+P
k-1
If [S] >> [E], d[ES]/dt = 0
Rate = k2[ES]
d[ES]/dt =0 is called steadystate condition.
d[ES]/dt = k1[E][S] –
k-1[ES] + k2 [ES] = 0
k1[E][S] = k-1[ES] + k2 [ES]
k1([E]T – [ES]) [S] = k-1[ES] + k2 [ES]
([E]T – [ES]) [S] / [ES] = (k-1 + k2) / k1 = KM
[E]T [S] – [ES] [S] = KM [ES]
[E]T [S] = [ES] (KM + [S])
[ES] = [E]T [S] / (KM + [S])
V = [ES] k2 Vmax = [E]T k2
V = Vmax [S] / (KM + [S]) Michaelis-Menten equation
when [S] = KM, V = ½ Vmax
Km = (k-1 + k2) / k1 , when k-1 >> k2 (rapid equilibrium), KM = KES = k-1/ k1
In the case of k-1 is comparable to k2 (Briggs-Haldane kinetics), KM = KES + k2 / k1
Lineweaver-Burk
double reciprocal
plot
Vmax / [E]T = turnover
number = kcat
kcat indicates catalytic
efficiency (kcat is larger,
reaction is faster)
KM indicates substrate
binding affinity (KM is
smaller,
binding is tighter)
kcat/Km is a measure of catalytic efficiency
vo =
kcat[E]T[S]
when [S]<<Km, little ES is formed, so [E] ~ [E]T ,vo = (kcat/Km) [E][S]
Km + [S]
kcat/Km is apparent second-order rate constant for an enzyme reaction,
It is smaller than diffusion-controlled limit 108~1010 M-1s-1
fK P
V
max
m
[P]eq
The Haldane Relationship: Keq =
=
VmaxrKmS
[S]eq
k1
The one-intermediate Model
Vmaxf
= k2[E]T
Vmaxr
= k-1[E]T
Competitive inhibitor
k1
k2
ES
E+S
k-1
+
I
KI
EI + S
E+S
No reaction
k-1
k2
EX
KMS
=
E+P
k-2
k-1 + k2
k1
E+P
Vo =
k2[E]T[S]
KM (1+
[I]
) + [S]
KI
KMP
=
K2 + k-1
K-2
Uncompetitive inhibitor
E+S
k1
k-1
KI’
ES
+
I
k2
ESI
E+P
Vmax[S]
Vo =
KM + (1+
No reaction
Mixed or Noncompetitive inhibitor
k2
k1
E+S
ES
E+P
k-1
+
+
I
I
KI
KI’
No reaction
ESI
EI
[I]
KI’
)[S]
Vmax[S]
Vo =
(1+
[I]
[I]
)KM + (1+ )[S]
KI’
KI
pH dependence of simple Michaelis-Menten Enzymes
E-
ES-
EH + S
KE1 H+
k1
k-1
ESH
KES1
EH2+
Vo =
H+
KES2
H+
KE2
k2
EH + P
H+
ESH2+
Vmax’[S]
KM’ + [S]
Vmax’ = Vmax/f2 KM’ = KM(f1/f2)
[H+]
k
f1 =
+ 1 + E2+
kE1
[H ]
f2 =
[H+]
+1+
kES1
kES2
[H+]
Bi-substrate Reactions
A+B
E
P+Q
E
Transfer Reaction
P-X + B
P + B-X
Terminology:
1. Substrates are designated by the letters A, B, C, and D in the order that
they add to the enzyme.
2. Products are designated P, Q, R, and S in the order that leave the enzyme.
3. Stable enzyme forms are designated E, F, and G with E being the free enzyme.
4. The numbers of reactants and products in a given reaction are specified, in
order, by the terms Uni (one), Bi (two), Ter (three), and Quad (four).
Types of Bi Bi reaction:
1. Sequential reactions (single-displacement), can be subclassifieid into
an Ordered mechanism (left) , and a Random mechanism (right).
A
E
A
B
k1 k-1
k2 k-2
EA
P
k3
EAB k-3 EPQ
P
B
Q
Q
k4 k-4
k5 k-5
EQ
E
E
EAB-EPQ
E
B
A
Q
P
2. Ping Pong Reactions
Ping Pong Bi Bi: double displacement
P
A
E
EA-FP
Q
B
F
FB-EQ
E
Rate equations
Ordered Bi Bi
KSAKSB
1
1
KMA
KMB
+
=
+
+
Vo
Vmax Vmax[A] Vmax[B] Vmax[A][B]
Rapid-equilibrium random Bi Bi
Ping Pong Bi Bi
1
1
=
Vo
Vmax
+
KSAKMB
VmaxKS
B[A]
1
1
KMA
KMB
+
=
+
Vo
Vmax Vmax[A] Vmax[B]
+
KMB
Vmax[B]
+
KSAKMB
Vmax[A][B]
slope = KMA/Vmax
Diagnostic plot for Ping Pong Bi Bi
1/vo
increasing
constant [B]
slope =
KM
A
intercept = 1/Vmax + KMB/Vmax[B]
KSAKMB
+ [B]
1/[A]
Vmax
double-reciprocal plots for a Ping Pong Bi Bi mechanism
1/vo
increasing
constant [B]
intercept = 1 + KMB/[B]
Vmax
Diagnostic plot for sequential Bi Bi
1/[A]
double-reciprocal plots for a Sequential Bi Bi mechanism
Differentiating random and ordered sequential mechanisms
1. Product inhibition:
2. isotope exchange
Enzyme reaction is complicated
1. Calculation of net rate constant
k1
A
k2
B
k-1
k3
C
k-2
k4
D
k-3
k5
E
k-4
F
The net rate constant for D -> E, k4’ = k4k5/(k-4 + k5)
The net rate constant for C -> D, k3’ = k3k4’/(k-3 + k4’)
…….etc
kP
k1’
P
A
F
The partitioning of A to F vs. P =k1’/kP
2. Use of transit times instead of rate constant
k1
k2
k3
k4
kn-1
EP1
EP2
EP3
EP4
…..
EPn
The total time from P1 to Pn, 1/k, is given by the sum of the transit times for each step
1/k = 1/k1 + 1/k2 + 1/k3 + 1/k4 + …. + 1/kn-1
As an example E + A
EA
E+P
The binding step is reduced to k1[A]k2 / (k-1 + k2)
[E]o/V = 1/k = (k-1 + k2) / k1[A]k2 + 1/k2
1/V = (k-1 + k2) / k1[A]Vmax + 1/Vmax
1/V = KM / [A] Vmax + 1/Vmax
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 establish
the kinetically preferred order of substrate addition and product release or
allow conclusions pertaining to the events occurring between substrate binding
and product release.
2. The value of kcat sets a lower limit on each of the first-order rate constants
governing the conversion of substrate to product following the initial collision
of substrate with enzyme. These include conformational changes in the enzymeSubstrate complex, chemical reactions (including the formation and breakdown
of 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 for
substrate 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 pathway
Including substrate binding, chemical reaction (substrate through intermediates to
product), and product release.
k2
k3
k4
k1
E+ S
ES
EX
EP
E+P
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 instrument
Measure 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 newly
formed cis double bonds.
UPPs synthesizes lipid carrier for bacterial cell wall assembly
Dolichyl pyrophosphate synthase catalyzes the lipid carrier for
Glycoprotein syntehsis
Enzyme single turnover rate is the same
with or without triton
10 mM E, 1 mM FPP, 50 mM [14C]IPP (With triton)
(Without triton)
kcat is 0.013 s-1 in the absence of triton and 190-fold higher (2.5 s-1) in the
presence of triton. However, the rate 2.5 s-1 under enzyme single turnover is
the same with or without triton
Pan et al., (2000) Biochemistry 10936-10942
UPPs single-turnover reaction time courses
10 mM UPPs, 1 mM FPP, 50 mM [14C]IPP
Y axis represents the sum of [14C]IPP
incorporated
10
Concentration (uM)
8
6
4
2
0
0
2
4
Time (sec)
6
8
10
1.2
The data represent the time courses of C20
(●), C25 (○), C30 (■), C35 (□), C40 (◆),
C45 (◊), C50 (▲), and C55 (△).
Concentration (uM)
1
0.8
0.6
0.4
0.2
0
-0.2
0
1
2
3
Time (sec)
4
5
6
The rate constants for IPP condensation
determined from single-turnover
fast
E + FPP
30 s
-1 -1
IPP 2 mM s
2.5 s-1
E-C20
E-FPP-IPP
E-FPP
-1
IPP
E-C30
3.5 s-1
E-C25-IPP
IPP
2.5 s-1
E-C30-IPP
E-C25
IPP
2 s-1
IPP
E-C35
E-C35-IPP
E-C20-IPP
3 s-1
E-C40
IPP
E-C50
3.5 s-1
3.5 s-1
E-C45-IPP
IPP
IPP
E-C50-IPP
E-C45
3 s-1
fast (with triton)
E-C55
E + C55
fast
E-C40-IPP
UPPs multiple-turnover reaction
0.75 mM enzyme, 6 mM FPP and
50 mM [14C]IPP without Triton
The data indicate formation of C55 (△),
C60 (●), C 65 (■), C70 (◆) and C75 (▲)
0.5
10
0.4
Concentration (uM)
Concentration (uM)
8
6
4
2
0.3
0.2
0.1
0
-0.1
0
0
20
40
60
80
Time (sec)
100
120
140
160
0
20
40
60
80
Time (sec)
100
120
140
160
Product dissociation is partially rate limiting and
protein conformational change is rate determining
0.001 s-1
E* + C55
E
0.4 s-1
E-C55
IPP (without triton)
E-C55-IPP
0.001 s
-1
-1
0.4 s
E-C60
E* + C60
E
0.5 s-1
IPP
E-C60-IPP
-1
E
0.001 s
E* + C65
-1
0.4 s
0.1 s-1
E-C65
IPP
E-C65-IPP
0.02 s-1
E-C70
0.001 s-1
E + C70 + C75
Substrate binding kinetics
E
k1[S]
ES
Rate = d[E]/dt = -k1[S][E]
d[E]/[E] = -k1[S]dt
ln([E]t / [E]o) = -k1[S]t
[E]t = [E]o exp (-k1[S]t)
[ES] = [E]o-[E]t = [E]o(1-exp (-k1[S]t))
kobs = k1 [S]
k1[S]
E
ES
kobs = k1[S] + k-1
k-1
The slope of kobs vs [S] gives kon and
intercept gives koff
Stopped-flow for measurements of protein-protein
and protein-small molecule interaction
B
A
Absorbance
Signal
Light
Flow Cell
Fluorescence
Signal
Stop Syringe
W91 has altered fluorescence upon FPP and IPP binding:
Use this property to measure kon
UPPs-FsPP + IPP
1 phase in 0.2 sec
Binding rates vs. [IPP] gives IPP
kon = 2 mM-1 s-1
Competition experiments to measure koff of FPP
using fluorescent substrate analog
OPP
O
PPO
O
PPO
O
OPP
OPP
CF3
Chen et al., (2002) J. Am. Chem. Soc. 124, 15217-15224
Substrate and product release rate
FPP is released at 30 s-1
fast
E + FPP
E-C25
E-C45
30 s-1
3.5 s-1
3.5 s-1
UPP is released at 0.5 s-1
-1 -1
IPP 2 mM s
2 s-1
2.5 s-1
E-C20
E-FPP-IPP
E-FPP
E-C30
E-C50
2.5 s-1
3 s-1
E-C35
E-C55
3 s-1
0.5 s-1
3.5 s-1
E-C40
E + C55
Can this method apply to drug-targeted prenyltransferases
to find non-competitive inhibitor?