enzymes are proteins with defined 3d structures ribonuclease a 2,3-dihydroxybiphenyl 1,2-dioxygenase...
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Enzymes are Proteins with Defined 3D Structures
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Ribonuclease A
2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)
-chymotrypsin
Active site:
uncatalysed reaction
acid-catalysed reaction
Freeenergy
Reaction co-ordinate
Eact(uncat)
Eact(cat)
ΔG
transition states
uncatalysed reaction
ESEPenzyme-catalysed
reaction
Freeenergy
Reaction co-ordinate
Eact(uncat)
Eact(cat)
ΔG
transition state
E.Int
A.
B.
S
P
SH+PH+
S
P
Enzyme Catalysis: What Enzymes Can & Can’t DoAcid-catalysed reaction
Enzyme-catalysed reaction
ESE + S
1. Direct UVP (UV active at 394 nm)
+ ENZ
time
slope v
S* P*2. Radiochemical
separate P*
scintillation counting
time
P*(counts perminute)
3. Indirect UV
ENZ ENZ
ENZ
UV active at 340 nm
+ ENZ
time
slope v
4. Coupled UV assay
ENZ ENZ 2
excess of coupling enzyme
monitor decrease in absorbance at 340 nm
S
A394
S P
NAD+NADH
A340
S P Q
NAD+NADH
Types of Enzyme Assay
1 Unit = activity required to convert 1 µmole S to P per minute
O
OUDPNHAc
HOHO
HN
ONH
O
HN
MurG
N-dansyl lipid I
O
NHAc
OO
OH
O
HN
O
NH-O2C
O
HN
O P
O
O-
O P
O-
O
O
O
NHAcHO
HO
OHN
NHO
NH
HN
O
NH
O
HN
CO2
O2C
S OO
NMe2
heptaprenyl
Ex 290 nm
340 nm
Em 500 nm
Fluorescence Resonance Energy Transfer Assay for MurG
0.2 M Tris pH 7.5, 10 mM MgCl2, 0.2% CHAPS2.7 µM Fl UDPGlcNAc, 3.0 µM dansyl lipid I+ 3.0 µg E. coli MurG
J.J. Li and T.D.H. Bugg,Chem. Commun., 182-183 (2004).
lyse cells at high pressure
extractcelldebris
high speedcentrifugation
Obtain or grow cellscontaining enzyme
enzymes
cell wall
Preparation of Cell Extract
Volume Enzyme activity Protein concentration Specific activity Purification
(ml) (units/ml) (mg/ml) (units/mg) (-fold)
Crude extract 14 13.1 62.0 0.212 1.0
DEAE sephadex pool 19 11.6 17.0 0.684 3.2
Phenyl agarose pool 11 11.8 0.085 140 662
MonoQ anion exchange pool 6.0 29.2 0.037 787 3710
Purification Table
Enzyme Purification
SDS-PAGE gel
k2
k-1
k1E + PESE + S
Michaelis-Menten Model for Enzyme Kinetics
Kinetic Model
= kcat [E]o [S]
Km + [S]+ [S]
k1
k2 [E]o [S]
k-1 + k2
Rate of production of P = k2 [ES] =
vmax
Km + [S]
vmax [S]=Observed rate v
where vmax = kcat [E0]
v
vmax/2
Km[S]
1/[S]
1/v
Lineweaver-Burk Plot:
1
vmax
Km= +
vmax
1
v
1
slope Km/vmax
1/vmax
Eadie-Hofstee Plot:
v = vmax - Km v
v
v/[S]
vmax
slope -Km
[S] [S]
Graphical Determination of Km & kcat
ES
E + S
[S] >> Km
v = kcat [Eo] Enzyme saturated with substrate
ESE + S
[S] = Km
v = kcat [Eo] Enzyme 50% saturated with substrate
2
ES
E + S
[S] << Km
v = kcat [Eo][S] Free enzyme + substrate
Km
What do Km & kcat really mean?
kcat - turnover number1st order rate constant (units s-1) for turnoverat high [S]
Km - Michaelis constantMeasure of affinity ofSubstrate bindingBUT not the same as Kd!
kcat/Km - catalytic efficiency2nd order rate constant(units M-1 s-1) for turnoverat low [S]
A) Competitive Inhibition
E + S ES E + P
EI
Ki + I
1/vmax
1/[S]
1/vinc. [I]
unchanged
Km(app) = Km 1 + [I]
Ki
B) Noncompetitive Inhibition
-1/Km
1/[S]
1/v
+ IKi
EI + S
E + PESE + S
EIS
Ki + I
inc. [I]
unchanged
Ki
vmax(app) = vmax 1 + [I]
Enzyme Inhibition - Reversible
OO
O OH
A
tRNA
O
NH3
NH2
OO
O OH
A
tRNA
O
NH3
O
NH3
L-Lys
D-Ala
γ-D-Glu
D-Ala
L-Ala
MurNAc
C55-OPP
NH
L-Lys
D-Ala
γ-D-Glu
D-Ala
L-Ala
MurNAc
C55-OPP
NH
L-Lys
D-Ala
γ-D-Glu
D-Ala
L-Ala
MurNAc
C55-OPP
GlcNAc GlcNAc GlcNAc
HOO
O R
N
PO
NH3
-O
N
N
N
NH2
R = OH, H
OO
O OH
tRNA
O
NH3
NH
L-Lys
D-Ala
γ-D-Glu
D-Ala
L-Ala
MurNAc
C55-OPP
GlcNAc N
N
N
N
NH2
Transition State Analogues for Ligase MurM
Inhibitor design: mimic tetrahedral transition state:
Transition state Phosphonate analogue
0.0 0.2 0.4 0.6 0.8 1.00
20
40
60
80
100
MurM activity (%)
Inhibitor Conc (mM)
Inhibition by 2’-deoxyadenosine analogue
IC50 = 100 µM
HOO
O
N
PO
NH3
-O
N
N
N
NH2
+ IKi
EI
E + PESE + S
kiE-I
Act
time
vi
1/vi
1/[I]
1/ki
slope Ki/ki
HN N
His57
O
Ser195
H
P
O
F
Enzyme Inhibition - Irreversible Inhibition
e.g. serine protease inhibitor DFP
Data SimulationData Simulation Single Exponential ModeSingle Exponential Mode
A = AA = A00 + +AA11 exp (- exp (-kk11t)t)
Double Exponential ModeDouble Exponential Mode
A= AA= A00 + + AA11 exp (- exp (-kk11t) + t) + AA22 exp (-exp (-kk22t)t)
Triple Exponential ModeTriple Exponential Mode
A= AA= A00 + + AA11 exp (- exp (-kk11t) + t) + AA22 exp (-exp (-kk22t) + t) +
AA33 exp (-exp (-kk33t) t)
Data SimulationData Simulation Single Exponential ModeSingle Exponential Mode
A = AA = A00 + +AA11 exp (- exp (-kk11t)t)
Double Exponential ModeDouble Exponential Mode
A= AA= A00 + + AA11 exp (- exp (-kk11t) + t) + AA22 exp (-exp (-kk22t)t)
Triple Exponential ModeTriple Exponential Mode
A= AA= A00 + + AA11 exp (- exp (-kk11t) + t) + AA22 exp (-exp (-kk22t) + t) +
AA33 exp (-exp (-kk33t) t)
Pre-Steady State Kinetics —— Application to C-C Hydrolase MhpC
CO2
O
O2C OH
CO2
O
O2C O
O2C OH CO2
CO2
-
-
-
-
+
Low pH A317 High pH A394
MhpC
-+
-
-
A270
0
1
0.4
0.6
0.8
1
1.2
1.4
0 0.2 0.4 0.6 0.8 1 1.2 1.4Association Time (s) -2? 10
Position (Arc s)
? 10-2
-4
-3
-2
-1
0
1
0 0.2 0.4 0.6 0.8 1 1.2 1.4Association Time (s) -2? 10
Error (Arc s)
? 10-1
0
1
2
0.5
1
1.5
0 0.2 0.4 0.6 0.8 1 1.2 1.4Association Time (s) -2? 10
Position (Arc s)
? 10-2
-5
-2.5
0
2.5
5
0 0.2 0.4 0.6 0.8 1 1.2 1.4Association Time (s) -2? 10
Error (Arc s)
Fit with single exponential (1 step) Fit with double exponential (2 step)
0.037-55.40.223-146H263A
18-117144-131Wild type.270nm(dienol P)
0.04078.5 0.34 96.6H263A
153.2145.6Wild type.317nm(dienol S)
kk2 2 (s(s-1-1))AA22 (×10 (×1033))kk1 1 (s(s-1-1))AA11 (×10 (×1033))pH=7.0
E + RFP E.RFP E.RFP E.succ E + succinate150s 18 s-1 -1
k
0.34s-1 0.04s-1
H263 is involved in both ketonization and C-C cleavage !H263 is involved in both ketonization and C-C cleavage !H263 is involved in both ketonization and C-C cleavage !H263 is involved in both ketonization and C-C cleavage !
Analysis of His263Ala Mutant
pH=8.0 KKMM (μM) (μM) kkcatcat (s (s-1-1)) kkcatcat/ K/ KM M (M(M
-1-1ss-1-1))
WT 6.8 28 4.1 x 106
H263A 5.5 0.0029 5.3 x 102
Kinetic ParametersKinetic Parameters Kinetic ParametersKinetic Parameters
Pre-steady state Kinetic ParametersPre-steady state Kinetic Parameters Pre-steady state Kinetic ParametersPre-steady state Kinetic Parameters
20ms 317nm20ms 317nm20ms 317nm20ms 317nm 200ms 317nm200ms 317nm200ms 317nm200ms 317nm 200s 317nm200s 317nm200s 317nm200s 317nm
Analysis of Ser110Ala Mutant
pH=8.0 KKMM (μM) (μM) kkcatcat (s (s-1-1)) kkcatcat/ K/ KM M (M(M
-1-1ss-1-1))
WT 6.8 28 4.1 x 106
S110A 18.5 0.0054 2.9 x 102
Kinetic ParametersKinetic Parameters Kinetic ParametersKinetic Parameters
Pre-steady state KineticPre-steady state Kinetic Pre-steady state KineticPre-steady state Kinetic
E + RFP E.RFP E.RFP E.succ E + succinate150s 18 s
-1 -1k
E.RFP*
140s-1 0.02s-1
3.1s-1
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