Title Identification of Lysyl and Glycyl Residues

Title
Author(s)
Identification of Lysyl and Glycyl Residues Located at the
Active Site in Glycogen Synthase and Their Roles in the
Catalytic Reaction
Furukawa, Koji
Citation
Issue Date
Text Version ETD
URL
http://doi.org/10.11501/3070477
DOI
10.11501/3070477
Rights
Osaka University
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48
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53
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戸、
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11A
ハリ
J
CHAPTERII
I
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r
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p
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l
a
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e
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p
t
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l
u
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da
g
a
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g
l
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F
i
g
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. Sequence
a
n
a
l
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i
soft
h
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t
r
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r
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r
o
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e
p
r
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s
e
n
t
sa
nu
n
i
d
e
n
t
i
f
i
e
d
aminoa
c
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d
. Thiss
t
r
u
c
t
u
r
ei
sc
o
n
s
i
s
t
e
n
twitht
h
eaminoa
c
i
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si
d
e
n
t
i
c
a
lt
ot
h
a
tfromMetl0t
oAsp21ex ∞ pt f
o
rLys15
(
T
a
b
l
e11) , andi
i
nt
h
ecompleteaminoa
c
i
ds
e
q
u
e
n
c
eo
fE
.c
o
l
iglycogens
y
n
t
h
a
s
e(
2
)
.
S
i
n
c
ea
nAP
2
P
L
l
a
b
e
l
e
dl
y
s
y
lr
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o
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o
s
i
t
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v
e
l
yi
d
e
n
t
i
f
i
e
d(7) ,
i
ti
sconcludedt
h
a
tLys15i
sl
a
b
e
l
e
dbyAP2-PL.
DISCUSSION
Ther
e
s
u
l
t
soft
h
ei
n
v
e
s
t
i
g
a
t
i
o
np
r
e
s
e
n
t
e
di
nt
h
i
sc
h
a
p
t
e
rprovide
e
v
i
d
e
n
c
ef
o
rt
h
ep
r
e
s
e
n
c
eofLys15a
tt
h
ea
c
t
i
v
es
i
t
ei
nE
.c
o/
ig
lycogen
h
eE-amino
s
y
n
t
h
a
s
e
. Basedont
h
egeometryoft
h
emodifyingreagent , t
groupofLys15i
sprobablyl
o
c
a
t
e
dc
l
o
s
et
ot
h
epyrophosphatemoietyof
.c
o
l
ienzyme , l
i
k
et
h
a
tof1, ys38 i
nt
h
er
a
b
b
i
t
ADP-glucosei
nt
h
eE
muscleenzymewhichi
sl
a
b
e
l
e
dbyUP2-PL(
4
)
.F
i
g
.7comparest
h
e
.c
o/
i(2) ,
a
m
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o
t
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r
m
i
n
a
ls
e
q
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e
n
c
e
samongglycogens
y
n
t
h
a
s
e
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r
a
b
b
i
tmuscle(5) , andhumanmuscle(
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.I
twasfoundt
h
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o
n
t
a
i
n
i
n
gt
h
e
Lys-X-Gly-Gly(Xr
e
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h
e
l
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e
dl
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s
y
lr
e
s
i
d
u
ei
sconservedi
nt
h
et
h
r
e
eenzymes , a
b
a
c
t
e
r
i
a
landmammalianmuscleenzymesa
r
en
o
thomologous(
3
)
. The
c
o
n
s
e
r
v
a
t
i
o
nofLys-X-Gly-Glyi
nt
h
et
h
r
e
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n
z
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r
n
e
smaybear
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f
l
e
c
t
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ont
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eimportanceoft
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e
q
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t
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m
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t
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n
edehydrogenases , aGly-Gly-X-Lyssequencei
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1
8
2
6
)
.P
i
s
z
k
i
e
w
i
c
ze
ta
.
l(
2
7
)repo 巾 d t
h
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o
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p
h
a
t
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o
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y
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y
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e
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ng
l
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t
a
m
a
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e
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y
d
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e
n
a
s
e
. Ont
h
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t
h
e
rhand , Lys38i
nr
a
b
b
i
tmuscleglycogen
s
y
n
t
h
a
s
ewasmodifieda
l
s
obyp
y
r
i
d
o
x
a
lphosphate , i
fahigher
c
o
n
c
e
n
t
r
a
t
i
o
noft
h
er
e
a
g
e
n
twasused(
5
)
. Ther e: fore , t
h
econservedl
y
s
y
l
r
e
s
i
d
u
e
si
nglycogens
y
n
t
h
a
s
e
sanddehydrogenasesseemt
obee
q
u
i
v
a
l
e
n.
t
Walkere
ta.
l(
2
8
)foundt
h
a
taGly-X-X申 X-X-Gly-Lys sequencei
s
conservedi
ns
e
v
e
r
a
lATP-andGTP-bindingp
r
o
t
e
i
n
swhichi
n
c
l
u
d
e
adenyl
a
t
ek
i
n
a
s
e(29) , H+-A
TPase(28) , andt
h
er
a
soncogeneproductp21
(
3
0
)
.I
thasbeendemonstratedt
h
a
tt
h
econservedl
y
s
y
lr
e
s
i
d
u
e
si
nt
h
e
s
e
p
r
o
t
e
i
n
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r
es
p
e
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i
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i
c
a
l
l
yl
a
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e
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d
e
n
o
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n
eorguanosine
h
emotifofal
y
s
y
lr
e
s
i
d
u
e
p
o
lyphosphopyωoxals (7 ,31-34). Therefore , t
i
nt
h
eg
l
y
c
i
n
e
r
i
c
hr
e
g
i
o
ni
sg
e
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r
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la
st
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r
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t
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r
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lelementof
p
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l
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p
h
a
t
e
b
i
n
d
i
n
gl
o
c
i
.
I
ti
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e
tf
u
l
l
yu
n
d
e
r
s
tandhowt
h
econservedsequencei
n
t
e
r
a
c
t
s
w
i
t
ht
h
epolyphosphatemoietyi
nt
h
eglycogens
y
n
t
h
a
s
e
. Onep
o
s
s
i
b
i
l
i
t
y
ct
switht
h
e
i
st
h
a
tt
h
econservedl
y
s
y
lr
e
s
i
d
u
ed
i
r
e
c
t
l
yi
n
t
e
r
a:
polyphosphatemoietyofs
u
b
s
t
r
a
t
es
u
g
a
rn
u
c
l
e
o
t
i
d
eandt
h
eg
l
y
c
y
l
1e
x
i
b
i
l
i
t
yi
nt
h
i
slocus , a
sobservedi
na
d
e
n
y
l
a
t
e
r
e
s
i
d
u
e
sprovidet
h
ef
k
i
n
a
s
e(
3
5
)
. Anotherp
o
s
s
i
b
i
l
i
t
yi
st
h
a
tt
h
eg
l
y
c
y
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d
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e
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h
e
m
s
e
l
v
e
s
i
n
t
e
r
a
c
td
i
r
e
c
t
l
ywitht
h
epolyphosphatem
o
i
e
t
y
.I
nt
r
i
o
s
ephosphate
isomerase , t
h
eamiden
i
t
r
o
g
e
n
soft
h
etwog
l
y
c
y
lr
e
s
i
d
u
e
s(Gly232and
Gly233)i
n
t
e
r
a
c
twitht
h
ephosphatemoietyofs山 strate t
r
i
o
s
ephosphate
i
n
c
et
h
i
ss
t
r
u
c
t
u
r
emaybea
p
p
l
i
c
a
b
l
eonlyt
o
(
3
6
)
. However , s
h
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l
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t
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k
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l
y
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u
n
d
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r
s
t
a
n
dt
h
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u
n
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t
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今''臼
i
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i
t
e
d
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r
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c
t
e
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u
t
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r
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t
h
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x
tchapter
-29-
TABLEI
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-38-
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i
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h
e
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i
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e
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l
e
c
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l
a
r
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.Maniatis , T. , Fritsch , E
Cloning~・ A
L
a
b
o
r
a
t
o
r
yManual, C
o
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dS
p
r
i
n
g}
'
I
a
r
b
o
rLaboratory ,
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r
i
n
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0
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t
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.Kuby , S
.A. , Palmieri , R
.H. , Frischat , A. , Fischer , A.H. , Wu , L
.
H. , Maland , L. , a
n
dManship , M.(
1
9
8
4
)B
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o
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h
e
m
i
s
t
r
y23 , 23932399
3
0
.Yuasa , Y. , Srivastava , S
.K. , Dunn , C
.Y. , Rhim , J
.S. , Reddy , E
.P
.
a
n
dAaronson , S
.A.(
1
9
8
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a
t
u
r
e303 , 775-779
-40-
CHAPTER1
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Roleoft
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e
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h
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l
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n
h
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t
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d
t
y
p
eandK15Qenzymesshoweds
i
m
i
l
a
rconvex
.
0:
tO
.
0
3andp
Kb= 10.7:tO.04 , andpKa= 6
.
5:
tO
.06
p
r
o
f
i
l
e
swithpKa= 6
e
s
p
e
c
t
i
v
e
l
y(
F
i
g
.3a) , s
u
g
g
e
s
t
i
n
ga
g
a
i
nt
h
a
tt
h
e
andpKb=9.6:tO.06 , r
i
o
n
i
z
a
t
i
o
ns
t
a
t
eoft
h
eε-amino groupofLys15i
snotd
i
r
e
c
t
l
yr
e
l
a
t
e
dt
ot
h
e
a
l
u
e
si
nt
h
eK15Qenzyme
c
a
t
a
l
y
t
i
cp
r
o
c
e
s
s
. Thes
l
i
g
h
ts
h
i
f
tofpKv
p
r
o
f
i
l
emaybeduet
oaminorchangeoft
h
ea
c
t
i
v
es
i
t
es
t
r
u
c
t
u
r
ecausedby
t
h
em
u
t
a
t
i
o
n
.
h
epKip
r
o
f
i
l
ef
o
rADPoft
h
eK15(2enzymewasq
u
i
t
e
1
ncontrast , t
d
i
f
f
e
r
e
n
tfromt
h
a
toft
h
ew
i
l
d
t
y
p
eenzyme(
F
i
g
.3
b
)
. Thew
i
l
d
t
y
p
e
i
v
i
n
gp
K
i
aandp
K
i
bvaluesof
enzymeshowedaconvexpKiprofile , g
:
t
O
.
0
6and10.1 :t O.03 , r
e
s
p
e
c
t
i
v
e
l
y
. Bya
n
a
l
y
z
i
n
gt
h
epKjp
r
o
f
i
l
eoft
h
e
7
.
0
-48-
wild 申 type
enzymea
td
i
f
f
e
r
e
n
ttempe凶 ures (
a
t20oCa
n
d30OC) , t
h
e
e
n
t
h
a
l
p
yc
h
a
n
g
e
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o
ri
o
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mol
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o
rt
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nt
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o
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h
e
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k
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l
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i
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e
. The 企Hjon v
a
l
u
eo
ft
h
el
a
t
t
e
rgroupi
si
ngooda
g
r
e
e
m
e
n
t
!mol)(17) , while
w
i
t
ht
h
ev
a
l
u
eo
fa
naminogroupi
np
r
o
t
e
i
n
s(
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al
t
h
esmall
企Hï on
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a
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ft
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eformergroupmayb
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r
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et
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e
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o
n
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r
o
t
o
n
a
t
i
o
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h
eﾟ
p
h
o
s
p
h
a
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egroup(pK.
.
6
.
3
)o
fADP. Ont
h
e
h
epKiv
a
l
u
e
sf
o
rADPo
ft
h
eK15Qenzymed
i
dn
o
tchange
o
t
h
e
rhand , t
i
napHr
a
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g
eo
f6
.
7
1
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4
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K
jp
r
o
f
i
l
ef
o
rAMPo
ft
h
ew
i
l
d
t
y
p
e
enzymewasa
l
s
oi
n
d
e
p
e
n
d
e
n
tonpH(
F
i
g
.3
b
)
. Theser
e
s
u
l
t
sc
l
e
a
r
l
yshow
fLys15w
i
t
hpKo
fa
b
o
u
t1
0i
sinvolved , i
ni
t
s
t
h
a
tt
h
eιamino groupo
nt
h
ei
o
n
i
ci
n
t
e
r
a
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t
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o
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t
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h
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h
o
s
p
h
a
t
egroupo
f
p
r
o
t
o
n
a
t
e
dform , i
ADP.
M
u
t
a
t
i
o
n0fGly17andGか 18 - Threem
u
t
a
n
tenzymesweret
h
e
n
nwhichGly17a
n
dGly18a
r
ei
n
d
i
v
i
d
u
a
l
l
yo
rs
i
m
u
l
t
a
n
e
o
u
s
l
y
prepared , i
r
e
p
l
a
c
e
dbyAla(
F
i
g
.2) , t
os
e
et
h
ee
f
f
e
c
to
fm
i
n
i
m
U
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h
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h
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l
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eGlym
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t
a
n
t
swere
p
u
r
i
f
i
e
dt
oh
o
m
o
g
e
n
e
i
t
ya
n
da
n
a
l
y
z
e
df
o
rt
h
e
i
rk
i
n
e
t
i
cp
a
r
a
m
e
t
e
r
s(
T
a
b
l
e
1
)
. Themostn
o
t
e
w
o
r
t
h
yc
h
a
n
g
ei
nk
i
n
e
t
i
cp
a
r
a
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e
t
e
r
swast
h
a
tt
h
ek ω
v
a
l
u
eo
ft
h
eG1
7
A enzymewast
h
r
e
e
o
r
d
e
r
so
fm
a
g
n
i
t
u
d
es
m
a
l
l
e
rt
h
a
n
h
o
u
g
ht
h
a
to
ft
h
eG18,Â. enzymewaso
n
l
y
t
h
a
to
ft
h
ew
i
l
d
t
y
p
eenzyme , t
. Ont
h
eo
t
h
e
rhand , c
h
a
n
g
e
si
nt
h
el(mv
a
l
u
e
sf
o
rADPｭ
3
.
2
f
o
l
ds
m
a
l
l
er
g
l
u
c
o
s
ea
n
dg
l
y
c
o
g
e
nweret
r
i
v
i
a.
l Thep
a
r
a
m
e
t
e
r
so
ft
h
ed
o
u
b
l
emutant
enzyme(G17NG18A)weres
i
m
i
l
a
rt
ot
h
o
s
eo
ft
h
eG17Aenzyme. CD
s
t
u
d
i
e
se
x
c
l
u
d
e
dap
o
s
s
i
b
i
l
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yt
h
a
tt
h
ec
o
n
f
o
r
m
a
t
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o
n
so
ft
h
eG1
7
Aa
n
d
G18Aenzymesa
r
eg
r
o
s
s
l
ya
l
t
e
r
e
d(
d
a
t
an
o
ts
h
o
w
n
)
. Theser
e
s
u
l
t
s
s
u
g
g
e
s
tt
h
a
tGly17p
l
a
y
sa
ni
m
p
o
r
t
a
n
tr
o
l
ei
nt
h
ec
a
t
a
l
y
t
i
cprocess , b
u
tn
o
t
i
nt
h
eb
i
n
d
i
n
go
fADP-glucose , w
h
i
l
et
h
er
o
l
eo
fGly18i
nt
h
ec
a
t
a
l
y
s
i
si
s
muchl
e
s
ss
i
g
n
i
f
i
c
a
n.
t
国 49
-
A
f
f
i
n
i
t
yL
a
b
e
l
i
n
gofGly17andGly18Mutants- Thea
f
f
i
n
i
t
yl
a
b
e
l
i
n
g
r
e
a
g
e
n
tAP
2
PLs
p
e
c
i
f
i
c
a
l
l
ymodifiedLys15i
nE
.c
o
l
iglycogensynthase
(Chapter1
1
)
. Ther
e
a
c
t
i
v
i
t
yofal
y
s
y
lr
e
s
i
d
u
ei
np
r
o
t
e
i
n
stowardt
h
e
formylgroupofp
y
r
i
d
o
x
a
lphosphateori
t
sd
e
r
i
v
a
t
i
v
e
sappearst
obe
dependentonpKoft
h
eaminogroup , whichi
nt
u
r
ni
sa
f
f
e
c
t
e
dbybothi
t
s
p
o
s
i
t
i
o
nandmicroenvironmenti
nt
h
ep
r
o
t
e
i
n(
1
1
)
. Onlyt
h
edeprotonated
formofanaminogroupi
sc
a
p
a
b
l
eofformingt
h
eS
c
h
i
f
fb
a
s
e
. Thep
o
s
i
t
i
o
n
oft
h
el
y
s
y
lr
e
s
i
d
u
er
e
l
a
t
i
v
et
ot
h
a
toft
h
eformylgroupi
nt
h
ebound
r
e
a
g
e
n
tmaya
l
s
oi
n
f
l
u
e
n
c
et
h
er
a
t
eoft
h
eS
c
h
i
f
fb
a
s
ef
o
r
m
a
t
i
o
n
. To
i
n
v
e
s
t
i
g
a
t
echangesi
nt
h
emicroenvironmentaroundt
h
eADP-glucosebindingLys15causedbyt
h
em
u
t
a
t
i
o
n
sofGly17andGly18 , t
h
ei
n
a
c
t
i
v
a
t
i
o
n
p
r
o
f
i
l
e
sbyp
y
r
i
d
o
x
a
lphosphateandi
t
sa
d
e
n
i
n
en
u
c
l
e
o
t
i
d
y
l
y
ld
e
r
i
v
a
t
i
v
e
sof
t
h
emutantenzymeswerecomparedwitht
h
o
s
eoft
h
ew
i
l
d
t
y
p
eenzyme. As
h
eG17A andG18Aenzymes , l
i
k
et
h
ew
i
l
d
t
y
p
eenzyme ,
showni
nF
i
g
.4 , t
werei
n
a
c
t
i
v
a
t
e
de
f
f
e
c
t
i
v
e
l
ybyAP2-PL. Thec
o
n
c
e
n
t
r
a
t
i
o
n
sofAP2-PL
ICso)were10 , 90 , and25μM fort
h
ew
i
l
d
ｭ
r
e
q
u
i
r
e
df
o
r50%i
n
a
c
t
i
v
a
t
i
o
n(
type , G17A , andG18Aenzymes , r
e
s
p
e
c
t
i
v
e
l
y
. Thed
i
f
f
e
r
e
n
c
e
si
nt
h
e1
C
s
o
1witht
h
e
v
a
l
u
e
soft
h
et
h
r
e
eenzymesf
o
rAP2-PLcorrespondedf
a
i
r
l
ywel
d
i
f
f
e
r
e
n
c
e
si
nt
h
e
i
rKmv
a
l
u
e
sf
o
rADP-glucose(
T
a
b
l
e1
)
. Interesti 時 ly ， t
h
e
w
i
l
d
t
y
p
eenzymewasi
n
a
c
t
i
v
a
t
e
dbyp
y
r
i
d
o
x
a
lphosphatewithanI
C
s
o
v
a
l
u
eof80μM ， whereast
h
eG17
A andG18Aenzymesweren
o
ti
n
a
c
t
i
v
a
t
e
d
byt
h
i
sr
e
a
g
e
n
tupt
o100μM. Ont
h
econtrary , t
h
ew
i
l
d
t
y
p
eenzymewas
h
eG17A andG18Aenzymeswere
n
o
ti
n
a
c
t
i
v
a
t
e
dbyAP3-PL , whereast
e
s
p
e
c
t
i
v
e
l
y
.
i
n
a
c
t
i
v
a
t
e
dbyt
h
i
sr
e
a
g
e
n
twithI
C
s
ov
a
l
u
e
sof150and60~tM ， r
Neithert
h
ew
i
l
d
t
y
p
enort
h
emutantenzymeswerei
n
a
c
t
i
v
a
t
e
dbyAP4-PL.
Theser
e
s
u
l
t
si
n
d
i
c
a
t
et
h
a
tb
o
t
hm
u
t
a
t
i
o
n
sofGly17andGly18s
i
g
n
i
f
i
c
a
n
t
l
y
change , i
nas
i
m
i
l
a
rway , t
h
er
e
a
c
t
i
v
i
t
yofLys15towardt
h
epyridoxal
derivatives , i
fi
ti
sl
a
b
e
l
e
da
l
s
oi
nt
h
emutante
n
z
y
r
n
e
s
.
-50-
Thus , l
a
b
e
l
e
dl
y
s
y
lr
e
s
i
d
u
e
(
s
)i
nt
h
eAP2-PL-orAP3-PL-modified
mutantenzyme(G17A)weret
h
e
nd
e
t
e
r
m
i
n
e
d
. TheG17
A enzyme
i
n
a
c
t
i
v
a
t
e
dwithAP2-PLo
rAP3-PLwasc
l
e
a
v
e
dwithtrypsin , andt
h
e
l
a
b
e
l
e
dp
e
p
t
i
d
ewasp
u
r
i
f
i
e
dbyHPLCa
sd
e
s
c
r
i
b
e
dunder"Experimental
P
r
o
c
e
d
u
r
e
s
"
. As
i
n
g
l
es
i
g
n
i
f
i
c
a
n
t
l
yf
l
u
o
r
e
s
c
e
n
tpeak , whoser
e
t
e
n
t
i
o
ntime
wasi
d
e
n
t
i
c
a
lwitht
h
a
toft
h
eAPγPL-labeled p
e
p
t
i
d
ed
e
r
ivedf
r
om t
h
e
nt
h
ee
l
u
t
i
o
np
r
o
f
i
l
e
sofbothmodified
w
i
l
d
t
y
p
eenzyme , wasobservedi
mutantenzymes(
F
i
g
.5
)
. Sequencea
n
a
l
y
s
i
sr
e
v
e
a
l
e
dt
h
a
tbothAP2-PL-and
AP3-PL-labeledp
e
p
t
i
d
e
ss
t
a
r
tfromMet1andLys15i
sa
c
t
u
a
l
l
yl
a
b
e
l
e
d
(
T
a
b
l
e1
1
)
.
DISCUSSION
Thes
t
u
d
yp
r
e
s
e
n
t
e
di
nt
h
i
sc
h
a
p
t
e
rh
a
sbeenundertakent
oe
l
u
c
i
d
a
t
e
y
s
X
G
l
y
.
G
l
y(Xr
e
p
r
e
s
e
n
t
sa
n
t
h
ef
u
n
c
t
i
o
n
a
lr
o
l
eofas
e
q
u
e
n
c
emotif, L
u
n
s
p
e
c
i
f
i
e
daminoa
c
i
dresidue) , conservedi
nt
h
enlammalianand
b
a
c
t
e
r
i
a
lglycogens
y
n
t
h
a
s
e
s(10) , a
l
t
h
o
u
g
ht
h
er
e
s
i
d
u
ecorrespondingt
o
Lys15oft
h
eE
.c
o
l
ienzymei
sArgi
nt
h
ey
e
a
s
tenzyme(
9
)
. Replacement
.c
o
l
ienzymebyGlno
rGlur
e
s
u
l
t
e
di
namarked
ofLys15oft
h
eE
a
l
u
e
sf
o
rADP-glucose , w
h
i
l
et
h
a
tbyArgl
e
a
dt
oonlya
i
n
c
r
e
a
s
ei
nKmv
6
f
o
l
di
n
c
r
e
a
s
e
.C
o
n
s
i
d
e
r
i
n
gt
h
ea
p
p
r
e
c
i
a
b
l
yh
i
g
ha
c
t
i
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andt
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o
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ew
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e
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h
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nt
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t
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n
t
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o
rA DP oft
,
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enzyme(
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g
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. Lossoft
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o
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t
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e
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o
r
o
fLys15byGlnorGlu , therefore , probablylowerst
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e
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e
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h
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h
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e
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. TheG17
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m
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r
witheacho
t
h
er
. ReplacementofGly17orGly18byAlamakesLys15
c
a
p
a
b
l
eofr
e
a
c
t
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n
gwithAP3-PL , b
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o
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r
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lphosphate(
F
i
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.
4
)
. Thismayr
e
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h
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o
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la
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h
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l
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.
However , t
h
i
schangemustbeverysllbtle , sin ∞
AP4-PL
r
e
a
c
t
swith
Lys15ofn
e
i
t
h
e
rt
h
ew
i
l
d
t
y
p
eenzymenorGly17amdGly18mutant
enzymes. Thef
a
c
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h
a
tr
e
p
l
a
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e
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t
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. Ont
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Enzyme
山一
TABLEI
K
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TABLE1
1
Thes
t
r
u
c
t
u
r
e
sofAP2-PL-andA
P
3
P
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Cycle
R
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Y
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1
2
3
4
5
6
7
8
9
10
1
1
12
1
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14
1
5
16
17
1
8
1
9
20
Met
Gln
Val
Leu
H
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s
Val
NDa
S
e
r
Glu
Met
Phe
Pro
Leu
Leu
NDb
Thr
Ala
Gly
Leu
Ala
248
138
102
136
55
84
88
28
1
1
3
75
6
5
50
70
1
8
43
56
53
1
9
Y
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l
d
pmol
Met
Gln
Val
Leu
H
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s
Val
ND
Ser
Glu
Met
Phe
Pro
Leu
Leu
ND
Thr
Ala
Gly
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Ala
118
150
106
105
22
72
34
47
28
65
54
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98
16
67
88
67
62
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. SequencesofprimersforPCRands
i
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mutagenesisofglycogensynthase. Them
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a
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r
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Time(
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FIG.5
. IsolationoftheAPn-PL-modifiedpeptidesi
nthewildｭ
typeandG17A enzymes. Thee
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今''u
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CHAPTERIV
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e
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l
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z
er
.
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i
t
e
d
i
r
e
c
t
e
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i
t
e
d
i
r
e
c
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e
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u
t
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e
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i
so
fLys277was
ta
l
.
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yat
w
o
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emethodo
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h
i
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t
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t
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i
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s
.
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RESULTS
I
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a
c
t
i
v
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t
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o
noftheK15QEnzymebyAP2-PL-Lys15i
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e
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o
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1
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e
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i
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g
efrom200t
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a
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t
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v
a
t
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o
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a
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t
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i
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e
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i
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o
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a
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1
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s
a
t
i
o
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l
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e
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i
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a
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. Onlyt
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o
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a
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n
a
c
t
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e
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n
. Thec
o
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t
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o
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u
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r
e
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o
r50%i
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a
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o
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b
o
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t500μM ， whichi
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a
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o
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l
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approximately3
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t
i
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st
h
a
tr
e
q
u
i
r
e
df
o
r50%i
n
a
c
t
y
p
eenzyme(Chapter1
1
)
. However , t
h
i
sd
i
f
f
e
r
e
n
c
ei
si
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a
l
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e
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o
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ed
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c
ei
nt
h
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h
a
p
t
e
r1
1
1
)
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c
o
n
c
e
n
t
r
a
t
i
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n
sofAP2-PLf
o
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h
ei
n
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en
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.
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b
s
t
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a
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t
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r
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t
i
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c
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f
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i
n
i
t
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o
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h
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nt
h
ew
i
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t
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1
1
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i
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h
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n
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c
t
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a
t
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o
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o
m
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yp
r
o
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e
c
t
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glucose , s
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e
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i
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i
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d
e
n
t
i
f
i
c
a
t
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et
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e
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o
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. Whent
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h
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i
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l
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e
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i
g
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t
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e
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h
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i
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c
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a
t
e
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g
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o
n
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e
n
t
r
a
t
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a
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y
s
i
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e
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e
a
l
e
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h
a
tt
h
es
t
r
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c
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l
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e
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t
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i
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d
.
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t
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t
u
r
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si
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e
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t
i
c
a
lt
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h
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e
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o
r
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o
l
iglycogens
y
n
t
h
a
s
e
Lys277i
nt
h
ecompleteaminoa
c
i
ds
e
q
u
e
n
c
eofE
(
2
)
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i
n
c
et
h
eAP2-PLl
a
b
e
l
e
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y
s
y
lr
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n
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o
tbep
o
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i
t
i
v
e
l
yi
d
e
n
t
i
f
i
e
d
(10) , i
twasconcludedt
h
a
tLys277i
ss
p
e
c
i
f
i
c
a
l
l
yl
a
b
e
l
e
dbyAP2-PL.
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e
a
c
t
i
v
i
t
i
e
sofLys277t
oAP2-, AP3-, andAP4
]
)
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a
c
t
i
v
i
t
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l
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e
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o
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i
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i
t
e
stowardt
h
e
n 川 eotidylyl
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e
r
i
v
a
t
i
v
e
sofpyωoxal varyi
nenzymes(
1
0
)
.I
na
d
e
n
y
l
a
t
e
kinase , Lys21i
sl
a
b
e
l
e
dbyanyofAP2-PL , AP3-PL , andAP4-PL(11 ,
1
2
)
.Ther
e
a
c
t
i
v
i
t
i
e
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nE
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l
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n
t
h
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et
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l
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a
t
e
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i
n
a
s
e
. Lys15i
nglycogens
y
n
t
h
a
s
ei
sl
a
b
e
l
e
dbyAP2-PL , b
u
tn
o
tbyAP3PLorAP4-PL(Chapter1
1
1
)
. OnlywhenGly17orGly18i
sr
e
p
l
a
c
e
dby
Ala , Lys15i
nt
h
emutantenzymesi
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a
b
e
l
e
dbyAP3-PLa
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e
l
la
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1
1
)
.I
twasi
n
t
e
r
e
s
t
e
di
nwhetherLys277i
sl
a
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l
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dbyAP3-
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i
g
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h
a
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h
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i
n
a
c
t
i
v
a
t
e
dbyn
e
i
t
h
e
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o1mM , t
w
i
c
ea
smucha
s
t
h
ec
o
n
c
e
n
t
r
a
t
i
o
nr
e
q
u
i
r
e
df
o
r50%i
n
a
c
t
i
v
a
t
i
o
nbyAP2-PL.
Sequencec
o
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l
p
a
r
ﾍ
s
o
n- Lys15l
a
b
e
l
e
dbyAP2-PLi
nt
h
em
o
d
i
f
i
c
a
t
i
o
n
oft
h
ew
i
l
d
t
y
p
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o
l
iglycogens
y
n
t
h
a
s
ei
sl
o
c
a
t
e
da
tt
h
et
e
t
r
a
p
e
p
t
i
d
e
sconserved ,
sequenceofLys-X-Gly-Gly , whichi
ex ∞pt
f
o
rX , i
nboth
l
t
h
o
u
g
ht
h
el
y
s
y
lr
e
s
i
d
u
e
mammalianandh
i
g
h
e
rp
l
a
n
tenzymes(3 , 13) , a
i
sr
e
p
l
a
c
e
dbya
na
r
g
i
n
y
lr
e
s
i
d
u
ei
nt
h
ey
e
a
s
te
n
z
y
r
n
e(
1
4
)
. Whent
h
e
sequencearoundLys277i
nE
.c
o
l
iglycogens
y
n
t
h
a
s
:
ewascomparedwith
t
h
eaminoa
c
i
ds
e
q
u
e
n
c
e
soft
h
emammalian(
3
)andy
e
a
s
tenzymes(14) ,
bothofwhichshownoo
v
e
r
a
l
ls
e
q
u
e
n
c
es
i
m
i
l
a
r
i
t
y:
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0t
h
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o
l
ienzyme ,
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i
m
i
l
a
rr
e
g
i
o
nwasf
o
u
n
d
. However , a
sshowni
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i
g
.4 , asequence
.c
o
l
ienzymei
sw
e
l
lconservedi
nmaizes
t
a
r
c
h
aroundLys277i
nt
h
eE
synthase , whichshowsa
b
o
u
t30%aminoa
c
i
di
d
e
n
t
i
t
yt
ot
h
eE
.c
o
l
i
enzyme(
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3
)
. Thisc
o
n
s
e
r
v
a
t
i
o
ns
u
g
g
e
s
t
st
h
a
tLys277i
nE
.c
o
l
iglycogen
s
.
s
y
n
t
h
a
s
ep
l
a
y
sa
ni
m
p
o
r
t
a
n
tr
o
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nenzymec
a
t
a
l
y
s
i:
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e
p
l
a
c
e
n
l
e
n
t01Lys277- Tor
e
v
e
a
lt
h
er
o
l
eofLys277 , t
h
eglycogen
s
y
n
t
h
a
s
emutantenzymei
nwhichLys277i
sr
e
p
l
a
c
e
dbyGlnwasprepared
v
i
as
i
t
e
d
i
r
e
c
t
e
dm
u
t
a
g
e
n
e
s
i
s
. Glnwaschosent
oexaminet
h
ee
f
f
e
c
tof
removalofap
o
s
i
t
i
v
echargea
tp
o
s
i
t
i
o
n277w
i
t
h
o
u
ts
i
g
n
i
f
i
c
a
n
tchangei
n
s
i
d
e
c
h
a
i
nb
u
l
k
i
n
e
s
s
. TableI
Isummarizest
h
ek
i
n
e
t
i
cp
r
o
p
e
r
t
i
e
soft
h
e
w
i
l
d
t
y
p
eandK277Qenzymes. TheKmv
a
l
u
e
sf
o
rADP-glucoseand
glycogenchangedl
i
t
t
l
e
.I
ncontrast , t
h
ek
c
a
tv
a
l
u
eoft
h
eK277Qenzyme
was140-timesa
ss
m
a
l
la
st
h
a
toft
h
ew
i
l
d
t
y
p
eenzyme. Theser
e
s
u
l
t
s
fhU
n
v
j
s
u
g
g
e
s
tt
h
a
tLys277i
si
n
v
o
l
v
e
di
nt
h
ec
a
t
a
l
y
t
i
cr
e
a
ct
i
o
nr
a
t
h
e
rt
h
a
n
b
i
n
d
i
n
gofADP-glucose.
D1SCUSS10N
Asd
e
s
c
r
i
b
e
di
nChapter11 , Lys15i
nE
.c
o
l
iglycogensynthasei
s
s
p
e
c
i
f
i
c
a
l
l
yl
a
b
e
l
e
dbyAP
2
PL. S
i
t
e
d
i
r
e
c
t
e
dm
u
t
a
g
e
n
e
s
i
ss
t
u
d
i
e
sshowed
t
h
a
tt
h
i
sl
y
s
y
lr
e
s
i
d
u
ei
smainlyi
n
v
o
l
v
e
di
nb
i
n
d
i
n
goft
h
es
u
b
s
t
r
a
t
eADPｭ
glucose , b
u
tn
o
te
s
s
e
n
t
i
a
lf
o
renzymea
c
t
i
v
i
t
y(Chapter1
1
1
)
. Obviously ,
aminoa
c
i
dr
e
s
i
d
u
e
(
s
)o
t
h
e
rt
h
a
nLys15a
r
ei
n
v
o
l
v
e
di
nt
h
ec
a
t
a
l
y
t
i
c
r
e
a
c
t
i
o
noft
h
i
senzyme. 1
nt
h
i
schapter , i
th
a
sbeenfoundt
h
a
tLys277i
n
t
h
eK15Qenzymei
ss
p
e
c
i
f
i
c
a
l
l
yl
a
b
e
l
e
dbyAP2-PL. Ther
e
s
u
l
t
sofs
i
t
e
ｭ
s
d
i
r
e
c
t
e
dm
u
t
a
g
e
n
e
s
i
shaved
e
m
o
n
s
t
r
a
t
e
dt
h
a
tt
h
i
sresidue , whichi
c
t
u
a
l
l
yp
l
a
y
sa
nimportant
conservedi
nmaizes
t
a
r
c
hs
y
n
t
h
a
s
e(2 , 13) , a
r
o
l
ei
nt
h
ec
a
t
a
l
y
t
i
cr
e
a
c
t
i
o
n
.
Onep
o
s
s
i
b
l
ee
x
p
l
a
n
a
t
i
o
nwhyLys277i
sn
o
tl
a
b
e
l
e
dbyAP2-PLi
nt
h
e
u
tl
a
b
e
l
e
di
nt
h
em
o
d
i
f
i
c
a
t
i
o
nof
m
o
d
i
f
i
c
a
t
i
o
noft
h
ew
i
l
d
t
y
p
eenzyme , b
t
h
eK15Qenzymei
st
h
a
tLys15i
sl
o
c
a
t
e
dc
l
o
s
et
ot
h
eformylgroupoft
h
e
s
l
a
b
e
l
i
n
gr
e
a
g
e
n
tboundt
ot
h
ew
i
l
d
t
y
p
eenzyme , whereasLys277i
. However , t
h
i
se
x
p
l
a
n
a
t
i
o
ne
n
c
o
u
n
t
e
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i
f
f
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c
u
l
t
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n
l
o
c
a
t
e
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l
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t
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v
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ar
i
k
eLys15i
n
i
n
t
e
r
p
r
e
t
i
n
gt
h
ef
i
n
d
i
n
gt
h
a
tLys277i
nt
h
eK15Qen :zyme , l
t
h
ew
i
l
d
t
y
p
eenzyme , i
sl
a
b
e
l
e
dbyAP2-PL , b
u
tn
o
l
tbyAP3-PLorAP4PL. Thel
a
b
e
l
i
n
gofLys277o
n
l
ybyAP2-PLi
nt
h
eK15Qenzymer
a
t
h
e
r
sw
e
l
la
sLys15 , i
sl
o
c
a
t
e
da
d
j
a
c
e
n
tt
ot
h
e
s
u
g
g
e
s
t
st
h
a
tLys277 , a
pyrophosphatemoietyoft
h
eI
a
b
e
l
i
n
greagent , a
l
t
h
o
u
g
hap
o
s
s
i
b
i
l
i
t
yt
h
a
t
mutationofLys15r
e
s
u
l
t
si
namovemento
fLys277c
l
o
s
et
ot
h
eformyl
groupoft
h
el
a
b
e
l
i
n
gr
e
a
g
e
n
tc
a
n
n
o
tbee
x
c
l
u
d
e
d
. Probablyt
h
er
e
a
c
t
i
v
i
t
y
ofLys15towardt
h
eformylgroupofAP2-PLi
smuchhighert
h
a
nt
h
a
tof
-70-
Lys277. OnceAP
2
-PLb
i
n
d
st
ot
h
emorer
e
a
c
t
i
v
eI ys15 , t
h
eADP.J
h
e
r
e
f
o
r
eLys277i
sn
o
tl
a
b
e
l
e
dbyt
h
esecond
bindings
i
t
ei
soccupied , t
.
moleculeoft
h
el
a
b
e
l
i
n
greagent
Themutantenzyme , i
nwhichLys277i
sr
e
p
l
a
c
e
dbyGln , was
preparedt
os
e
et
h
ee
f
f
e
c
tofremovaloft
h
ep
o
s
i
t
i
v
echargea
tt
h
ep
o
s
i
t
i
o
n
ofLys277withaminimumchangeoft
h
es
i
d
e
c
h
a
i
nb
u
l
k
i
n
e
s
sont
h
e
enzymer
e
a
c
t
i
o
n
. Thek
i
n
e
t
i
cp
a
r
a
m
e
t
e
r
soft
h
emutantenzymeshowst
h
e
p
o
s
i
t
i
v
echargeoft
h
i
sr
e
s
i
d
u
ei
sd
i
s
p
e
n
s
a
b
l
ef
o
rt
h
eb
i
n
d
i
n
goft
h
e
s
u
b
s
t
r
a
t
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m
p
o
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a
n
tf
o
rt
h
ec
a
t
a
l
y
t
i
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e
a
c
t
i
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. Ther
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eEｭ
aminogroupofLys277i
nt
h
ec
a
t
a
l
y
t
i
cp
r
o
c
e
s
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i
s
c
u
s
s
e
di
nt
h
e
.
f
o
l
l
o
w
i
n
gchapter
ー
弓/
A
』
TABLEI
Sequenceo[s
y
n
t
h
e
t
i
cprimers[ors
i
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e
d
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r
e
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t
e
dmutαgenesis
Primer
Sequence
275
280
-Ala-Glu-Asn-Lys-Arg-Gln-LeuGCG GAA AAT AAG CGC CAG TTA
CGC CTT TTA TTC GCG GTC AAT
*
277Q+
5'-GCG GAA AAT CAG CGC CAG T-3'
277Q-
3'-CGC CTT TTA GTC GCG GTC A-5'
4同
641+
1
1
0
9
-
5' ー CATCCAATTGCCATGGTCAT-3'
3'-CCACGACGTCCTTCCAAAG-5'
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o
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a
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e
r
i
s
k
s
.
今ん
内/
TABLE1
1
K
i
n
e
t
i
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r
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20
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Time(
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f
f
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c
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F
i
g
.1
. InactivationofK15Qenzymebyj\P2 ・ PL. a , e
t
h
ec
o
n
c
e
n
t
r
a
t
i
o
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h
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e
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l
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r
o
p
h
o
s
p
h
a
t
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PH 7.0) , 2m Mdithiothreitol ,
10%glycerol , 0.6μM K15Qenzyme , andAP2-PLa
t200μM (0 ) , 400
μM( ム )， 600μM( 口)， o
r800μM( ・). Them
i
x
t
u
r
ewasi
n
c
u
b
a
t
e
da
t20
o
cforvarioustimes , and25μ1 waswithdrawn , andmixedwith1.5μ1 of
30m Msodiumb
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y
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i
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e
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f
t
e
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h
eenzymea
c
t
i
v
i
t
ywas
measured. b , e
f
f
e
c
tofs
u
b
s
t
r
a
t
e
sont
h
ei
n
a
c
t
i
v
a
t
i
o
n
. K15Qenzymewas
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n
c
u
b
a
t
e
dwith800μM AP
2
-PLi
nt
h
ep
r
e
s
e
n
c
eo
f20m MADP-glucose
(0 ) , 20 m MADP( ム )， o
rnone(・). Otherc
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n
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i
t
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o
n
sweree
s
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n
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r
o
f
i
l
e
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r
y
p
s
i
nd
i
g
e
s
t
softheAP2 ・ PL­
labeledK15Qenzyme. Thet
r
y
p
s
i
nd
i
g
e
s
to
fK15Qenzymewhichwas
l
a
b
e
l
e
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nt
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ea
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n
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h
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r
e
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n
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e
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t
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n
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h
ef
l
u
o
r
e
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e
n
c
e(
e
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c
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o
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i
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>
60
40
+
t
J
<
~
20
。
。
200
400
600
800
1000
Reagent(μM)
F
i
g
.
3
. InactivationofK15Qenzymeby}
¥
P
n
P
L
. TheK15Q
enzymewasi
n
c
u
b
a
t
e
dw
i
t
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a
r
i
o
u
sc
o
n
c
e
n
t
r
a
t
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o
n
so
fAP2-PL(0) , AP3PL(ム)， o
rAP4-PL(口) a
t20oCf
o
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borohydidea
t0oCf
o
r10m
i
n
. Ther
e
s
i
d
u
a
la
c
t
i
v
i
t
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h
el
a
b
e
l
e
d
enzymewasmeasureda
f
t
e
rd
i
l
u
t
i
o
n
.
-76-
280
270
E
.c
o/
ig
lycogensynthase
DT-LEDKAEN 園 RQSQ 工 AMGS
女
maizes
t
a
r
c
hsynthase
300
*
・大・台
女*
310
セ
セ
STAVEAKALNKEALQAEVGL
F
i
g.4
. Comparisonoft
h
eaminoa
c
i
dsequencesbetweenE
.
c
o
l
iglycogensynthaseandmaizestarchsynthase. Gaps
(
h
y
p
h
e
n
s
)werei
n
t
r
o
d
u
c
e
df
o
rmaximumm
a
t
c
h
i
n
g
. Numbersabove
s
e
q
u
e
n
c
e
sa
r
er
e
s
i
d
u
enumbersi
ne
a
c
he
n
z
y
m
e
.As・ terisks i
n
d
i
c
a
t
e
i
d
e
n
t
i
c
a
lr
e
s
i
d
u
e
s
. Thel
y
s
y
lr
e
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i
d
u
el
a
b
e
l
e
db
yAP2-PLi
sboxed.
国 77
-
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1
3
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. Frakes , I., Hardy , T
(1990) よ Bio l.
Chem.265 , 20879-20886
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-86-
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