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20 Φεβ 2013 (πριν από 4 χρόνια και 6 μήνες)

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Coulomb interactions between internal
ionizable groups and surface residues

Victor Khangulov

May 13, 2009


Institute in Multiscale Modeling of Biological interactions

Johns Hopkins University


Laboratory of Dr. Garcia
-
Moreno


2

Many biochemical processes are
governed by internal ionizable groups


Photoactivation


Ion homeostasis


H
+

transport


e
-

transfer


Catalysis

Most ionizable residues are
located on the protein surface.

Internal ionizable residues
are responsible all forms of
energy transduction:

3

Many industrial processes require altered
pH dependent properties of enzymes


Starch liquefaction (production of ethanol and high
-
fructose syrup)


α
-
amylase is not active below pH 6.


Addition of salts is needed to adjust its activity to lower pH.

[Andrew Shaw, Richard Bott, and Anthony Day Current Opinion in Biotechnology 10 (4), 349 (1999)]



Dye bleaching/Detergents


Fungal peroxidase is not active at higher pH (~10) and high
peroxide concentration.


Directed evolution is necessary to increase its activity.

[Joel Cherry et al. Nature biotechnology 17 (4), 379
-
84 (Apr 1999) ]



Feed additives (digestion of phosphorous as phytate)


Phytase: very low activity at lower pH (3.5) of the stomach.

[Taewan Kim et al. Applied and Environmental Microbiology 72 (6), 4397 (2006)]


Interactions between surface and active site ionizable groups
can be modified to change pH profile of enzyme activity.

4

Internal lysines are crucial in catalysis


Acetoacetate decarboxylase



catalyzes formation of acetone
and CO2 from acetoacetate.



Mandelate racemase



catalyzes
equilibration of (R)
-

and (S)
-
enaintioners of mandelate.

Lys
-
115 p
K
a

= 6.0

Lys
-
166 p
K
a

= 6.4

Bharati Mitra et al.

Biochemistry 34 (9), 2777 (1995)

Lane Highbarger and John Gerlt

Biochemistry 35 (1), 41 (1996

5

Apparent p
K
a

values of Lys at 25
internal positions

Normal p
K
a

of Lys in water

6

p
K
a

Determination: Ideal Case

Titration of Lys
-
25 in

L25K



G
p
H



G
c

R
T
l
n
1

e
z
2
.
3
0
3
p
H

p
K
a
D




1

e
z
2
.
3
0
3
p
H

p
K
a
N




Background

L25K

Assumption:
Group behaves independently




We know p
K
a

values of all H,D, and E!




His
-
8 p
K
a

= 6.3



His
-
121 p
K
a

= 5.4



Asp
-
21 p
K
a

= 6.5



Everything else titrates

4.5




For “ideal” cases, H8, H121 and D21 are
not affected by internal lysine.


7

p
K
a

Determination: Non
-
Ideal Case

Titration of Lys
-
62 inT62K



G
p
H



G
c

R
T
l
n
1

e
z
2
.
3
0
3
p
H

p
K
a
,
1
D




1

e
z
2
.
3
0
3
p
H

p
K
a
,
1
N





R
T
l
n
1

e
z
2
.
3
0
3
p
H

p
K
a
,
2
D




1

e
z
2
.
3
0
3
p
H

p
K
a
,
2
N




Background

T62K

His
-
8, His
-
121 or Asp
-
21 are

Affected by the ionization of Lys
-
62


8

p
K
a

of Lys
-
62 shifts down in D21N variant

T62K

Background

D21N/T62K

Background

p
K
a

= 8.1
±

0.1

p
K
a

= 7.0
±

0.2

9

Titration of Asp
-
21 shows dependence
on the presence of Lys
-
62

Asp
-
21 in ∆+PHS

p
K
a

= 6.6
±

0.1

n = 2.0
±

0.02

Asp
-
21 in T62K

p
K
a

≈ 4.3
±

0.5

n = 0.6
±

0.03

10

Titration of other groups in the
presence of Lys
-
62

D21

D19

E43

E67

∆+PHS

T62K

T62K

∆+PHS

∆+PHS

T62K

T62K

∆+PHS

11

Effect of Lys
-
62 on the p
K
a

of

Asp
-
21

∆G
ij

= 1.36 (p
K
a
2



p
K
a
1
)


= 1.36 (6.6


4.3) =
3.0 kcal/mol

p
K
a
2
= 6.6

∆+PHS

T62K

D21N

D21N/T62K

p
K
a
1
= 4.3

12

NMR confirms p
K
a

of Lys
-
62
obtained through linkage analysis

p
K
a

= 8.1
±

0.02

(Linkage p
K
a

= 8.1
±

0.1
)

T62K

Global fit of 1H amide chemical shift

13

Lys
-
62 p
K
a

shifts further down in
D21N variant

p
K
a

= 6.7
±

0.03

(Linkage p
K
a

= 7.0
±

0.2
)


D21N/T62K

14

Effect of Asp
-
21 on p
K
a

of Lys
-
62

∆G
ij

= 1.36 (p
K
a
2



p
K
a
1
)


= 1.36 (8.1


6.7 ) =
1.9 kcal/mol

p
K
a
2
= 8.1

∆+PHS

T62K

D21N

D21N/T62K

p
K
a
1
= 6.7

15

∆G
ij

is not symmetric between

Asp
-
21 and Lys
-
62


∆G
ij

(Lys
-
62) =
1.9 kcal/mol

∆Gij (Asp
-
21) =
3.0 kcal/mol

p
K
a
2
= 8.1

∆+PHS

T62K

D21N

D21N/T62K

p
K
a
1
= 6.7

p
K
a
2
= 6.6

p
K
a
1
= 4.3

This is the best
estimate of
∆G
ij

16

The magnitude of the coupling between internal
and surface ionizable groups could be governed
by other factors

Electronic
polarization

Global
unfolding

Local
unfolding

Relaxation of
permanent
dipoles

Water
penetration

Bulk water

Fixed
permanent
dipoles

Second internal
charge

Surface
charges

Fluctuations
of surface
charges

17

T62K structure with Lys
-
62 in neutral state

Background

T62K

18

Isolated regions exhibit large changes in
1
H
N

chemical shift upon titration of Lys
-
62 (pH 7
-
9)

Asp
-
21
region

Lys
-
62
region

19

Summary


There appears to be significant interaction
between Lys
-
62 and Asp
-
21.



Titration of Lys
-
62 does not induce major
structural reorganizations.



Ionization of Lys
-
62 induces minor structural
reorganizations.



The Coulomb interaction between Asp
-
21 and
Lys
-
62 is 1.9 kcal/mol.


20

9 Internal Lys Variants Show Evidence of
Coupling to another ionizable Residue

T62K

21

Crystal structures where internal Lys is
coupled to the ionization of Asp
-
21

T62K

V104K

9
Å

6
Å

22

Future experiments:


What are the structural consequences of
ionization of Lys
-
62?




Is the phenomenon observed in T62K general?
(study 8 Lys variants that exhibit coupling to
another residue).


23

Progress on x
-
ray structures


Mutation

Background

Side
-
chain

RES (
Å)

pK
a

pH

r
ij
(
Å)

None

∆+PHS

n/a

1.85

n/a

8.0

n/a

V23K

PHS

2.0

7.3

7.0

8.0

L36K

∆+PHS

Neutral

1.90

6.2

8.4

6.4

T62K

PHS

Polar

2.10

8.1

8.0

6.1

L103K

∆+PHS

Polar

2.0

8.2

7.0

3.9

V104K

∆+PHS

Polar

2.0

7.8

7.0

8.9

D21N/T41K

∆+PHS

1.90

?

8.5

F34K

T41K

A90K

A109K

24

His
-
8 and His
-
121 p
K
a

values show no
dependence on the presence of Lys
-
62



obs
(
pH
)


AH


A


10
n

(
pH

pK
a
)
1

10
n

(
pH

pK
a
)
25

Simulation of interactions between
two ionizable groups

Asp
-
21 p
K
a

= 5.0

Lys
-
62 p
K
a

= 8.1

Asp
-
21 p
K
a

= 6.5

Lys
-
36 p
K
a

= 7.2

26

Relationship between Gibbs

free energy and a shift in p
K
a

value

+

1
.
3
6
k
c
a
l
m
o
l
p
H

1
27

The free energy of ionization of an
internal group can be calculated from its
p
K
a

shift

1
.
3
6

p
K
a

G
i
o
n

28

Primary energetic contributions

to the p
K
a

value of internal groups

3
3
2
Z
i
2
2
r
i
o
n
1

p

1

w






Always
unfavorable

p
K
a

of Glu




If unfavorable,

p
K
a

of Glu



If favorable,

p
K
a

of Glu





G
B
o
r
n
,
i


G
C
o
u
l
o
m
b
,
i

3
3
2
Z
i
Z
j

e
f
f
r
i
j
i

j

29

Modified Hill Equation

)
(
)
(
)
(
)
(
2
2
1
1
2
1
1
10
10
1
10
10
)
(
a
a
a
a
a
a
pK
pK
pH
n
pK
pH
pK
pK
pH
n
A
pK
pH
AH
AH
obs
pH




















2
-
site

1
-
site

))
(
(
))
(
(
1
10
1
10
)
(
a
a
pK
pH
n
pK
pH
n
AH
AH
obs
pH









30

Surface mutations affect enzyme
catalysis


Serine protease

Russell, A. J., and Fersht, A. R. (1987)
Nature

328
(6130), 496
-
500

Jackson, S. E., and Fersht, A. R. (1993)
Biochemistry

32
(50), 13909
-
13916



Catalytic His
-
64 interacts with surface Asp
-
99 (13 Å away) and
Glu
-
156 (15 Å away)


∆p
K
a

≈ 0.4, ∆G ≈ 0.6 kcal/mol



Thermolysin
-
like protease


de Kreij, A., van den Burg, B., Venema, G., Vriend, G., Eijsink, V. G., and Nielsen, J. E. (2002)
J
Biol Chem

277
(18), 15432
-
15438




Catalytic Glu
-
143 and His
-
231 interacts with carboxylic groups
10
-
15
Å away.


Max ∆p
K
a

≈ 0.5, ∆G ≈ 0.6 kcal/mol