Glycogen Phosphorylase - California State University

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Transcript Glycogen Phosphorylase - California State University

Glycogen Phosphorylase
Carlos Mendoza
Lee Sutton
Kristen Harris
• Glycogen Phosphorylase is an enzyme found in glycogen
granules in the cytosol
• Glycogen Phosphorylase releases sugar from glycogen by
clipping off glucose from the chains of a glycogen
molecule. It catalyzes a phosphorolysis reaction where it
adds a phosphate to break off glucose:
(glucose)n + Pi <<====>> glucose 1-phosphate + (glucose)n-1
Protein Characteristics
1
61
121
181
241
301
361
421
481
541
601
661
721
781
kqisvrglag
qqhyyekdpk
lgngglgrla
karpeftlpv
dfnlkdfnvg
fksskfgcrd
tcaytnhtvi
veegavkrin
wlvlcnpgla
reykvhinpn
pgyhmakmii
teasgtgnmk
yydripelrq
yknprewtrm
venvtelkkn
riyylslefy
acfldsmatl
hfygrvehts
gyiqavldrn
pvrtnfdafp
pealerwpvh
mahlciagsh
eiiaerigee
slfdvqvkri
klitaigdvv
fmlngaltig
iieqlssgff
virniatsgk
fnrhlhftlv
mgrtlqntmv
glaaygygir
qgakwvdtqv
laenisrvly
dkvaiqlndt
lletllprhl
avngvarihs
yisdldqlrk
heykrqllnc
nhdpvvgdrl
tmdganvema
spkqpdlfkd
fssdrtiaqy
kdrnvatprd
nlalenacde
yefgifnqki
vlampydtpv
pndnffegke
hpslaipelm
qiiyeinqrf
eilkktifkd
llsyvddeaf
lhvitlynri
rviflenyrv
eeageenffi
ivnmlmhhdr
areiwgveps
yyfalahtvr
atyqlgldme
cggwqmeead
pgyrnnvvnt
lrlkqeyfvv
rvlvdlerld
lnrvaaafpg
fyelephkfq
irdvakvkqe
kkepnkfvvp
slaekvipaa
fgmrvedvdr
fkvfadyeey
rqrlpapdea
dhlvgrwirt
eleeieedag
dwlrygnpwe
mrlwsakapn
aatlqdiirr
wdkawevtvk
dvdrlrrmsl
nktngitprr
nklkfaayle
rtvmiggkaa
dlseqistag
ldqrgynaqe
vkcqervsal
ap
8GPB Amino acid chain
• sizeof(Glycogen Phosphorylase) = 832 aa
In humans found on chromosome 20
• Glycogen Phosphorylase can be in a dimer or tetramer
form. However, it is thought to be mostly in the dimer
form in vivo. Each monomer consists of two domains:
• C terminal domain - domain responsible for catalysis of the
reaction (blue)
• N terminal domain - domain responsible for regulation of the
enzyme (pink)
Both domains consist of a beta sheet core surrounded by
layers of alpha helical segments
C TERMINAL DOMAIN
• The active site is actually in the cleft between the
C and N terminal domains
• A cofactor PLP (pyridoxal phosphate) binds near
the active site to facilitate the reaction
N TERMINAL DOMAIN
• Contains different allosteric effector sites
that determine when the enzyme is active (R
state) or inactive (T state.)
• Can be phosphorylated at Serine 14 to induce
active state (see previous slide)
• Or can bind with Amp to induce active state
Change from T to R state
• Problem - T state is found as a monomer, but R
state only crystallized as a tetramer.
• Glycogen Phosphorylase forms tetramers when
there is a lot of the enzyme present, which is the
case when glucose is needed and there is a lot of
the R state of the enzyme present.
• This makes the two hard to compare visually.
Upon phosphorylation • Amino acids 10-22 swing 120 degrees
causing a change in both the N and C
domain overall structures
The phosphate group is in red, residues 10-22 surround it
Additional change from T to R
State
• Residues 280-286 go from blocking active
site in T state to pulled out away from it in
the R state.
AMINO ACIDS 280-286 IN YELLOW
T (INACTIVE)
R (ACTIVE)
• MAIN POINT IS THAT BINDING AT
EFFECTOR SITES FOR PHOPSHATE OR
AMP LEAD TO CHANGES IN THE
PROTEIN’S SHAPE THAT MAKES IT
MORE ABLE TO ACCEPT GLYCOGEN
AT IT’S ACTIVE SITE
Phylogenetic trees
unrooted
Rooted(from unrooted tree)
Tree characteristics
•All sequences are closely conserved
•Bacteria tend to be more closely related to other
bacteria than to the others
•The brain, liver, and muscle forms of Glycogen
phosphorylase tend to group together
•The fruit fly is grouped closer to the vertebrates
than to the bacteria as expected
•The brain, liver, and muscle forms in one organism
are almost identical to the brain, liver, and muscle
forms in different organisms
•Glycogen phosphorylase showed up in a lot of
bacteria, and then in vertebrates, but not much in
the middle this might be because it hasn’t been
studied much in other organisms
Evolution of Glycogen phosphorylase
•Over time glycogen phosphorylase has changed
very little it is very similar in bacteria and
vertebrates
•There is more variation in bacteria glycogen
phosphorylase than in vertebrates
•This might be to the fact that bacteria has been
around much longer and has had more time to
change
•Or it might be that Glycogen phosphorylase in
vertebrates is unable to change with out causing
major problems changes in bacteria are not as
damaging
Hydrophobicity
•Exist in a hydrophilic environment
•The percentage of solvent-accessible hydrophobic
elements is much greater than in the “average”
globular protein
Hydrophobicity
Grease
PDB entry 8GPB
Blast
•Not much was returned besides glycogen
phosphorylase
•A few other enzymes that break down sugars
were returned such as maltodextrin phosphorylase
and Glucan phosphorylase
Software Tools
• Structure Visualization : Cn3D software, using
•
•
•
•
images from NCBI.
Phylogenetic Analysis : Used ClustalW on the
Biology Workbench
Hydrophobicity Analysis: Used Grease on the
Biology Workbench
BlastP used in biology Workbench to search for
similar proteins
Used Google for everything
References
Fletterick, Robert and Stephen Sprang. Glycogen phosphorylase Structures and
Function.Department of Biochemistry and Biophysics, School of Medicine,
University of California. Acc. Chem Res. 1982, 15, 361-369.
Goodsell, David. Glycogen Phosphorylase. Molecule of the Month. Protein Data
Bank. Internet. http://www.rcsb.org/pdb/molecules/pdb24_1.html. 5/13/03.
Gillis, Jeremy. Glycogen Phosphorylase - Structure and Function Term Paper.
Internet.
http://www.chem.uwec.edu/webpapers_F98/gillis/Structure/struct_descrip.htm
l. 5/13/03.