Download Enzyme Mechanisms

Transport & Signaling;
Nucleic Acid Chemistry
Andy Howard
Introductory Biochemistry
30 September 2008
Biochemistry:Transport; Nucleic Acids
09/30/08
What we’ll discuss

Membrane transport

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Energetics
Active transport
Transporting big
molecules
Membrane Signaling

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Nucleic acid chemistry

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
Adenylyl cyclase
Inositol-phospholipid
signaling pathway
Receptor tyr kinases
Pyrimidines: C, U, T
Purines: A, G
Nucleosides
09/30/08 Biochemistry:Transport; Nucleic Acids
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Protein-facilitated
passive transport

All involve negative DGtransport

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Uniport: 1 solute across
Symport: 2 solutes, same direction
Antiport: 2 solutes, opposite directions
Proteins that facilitate this are like
enzymes in that they speed up
reactions that would take place slowly
anyhow
These proteins can be inhibited,
reversibly or irreversibly
09/30/08 Biochemistry:Transport; Nucleic Acids
Diagram courtesy
Saint-Boniface U.
p. 3 of 50
Kinetics of passive transport



Michaelis-Menten saturation kinetics:
v0 = Vmax[S]out/(Ktr + [S]out) …
we’ll revisit this after we do enzyme
kinetics
Vmax is velocity achieved with fully
saturated transporter
Ktr is analogous to Michaelis constant:
it’s the [S]out value for which half-maximal
velocity is achieved.
09/30/08 Biochemistry:Transport; Nucleic Acids
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Primary active
transport


Energy source:
usually ATP or light
Energy source directly
contributes to overcoming
concentration gradient


QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Bacteriorhodopsin
PDB 1F50, 1.7Å
25 kDa monomer
Bacteriorhodopsin: light energy used to drive
protons against concentration and charge
gradient to enable ATP production
P-glycoprotein: ATP-driven active transport of
many nasties out of the cell
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Secondary active
transport

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Active transport of one solute is
coupled to passive transport of
another
Net energetics is (just barely)
favorable
Generally involves antiport


QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Pyrococcus Multisugar transporter
PDB 1VCI
83 kDa dimer
Bacterial lactose influx driven by
proton efflux
Sodium gradient often used in
animals
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Complex case:
Na+/K+ pump


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Typically [Kin] = 140mM, [Kout] = 5mM,
[Nain] = 10 mM, [Naout] = 145mM.
ATP-driven transporter:
3 Na+ out for 2 K+ in
per molecule of ATP hydrolyzed
3Na out: 3*6.9 kJmol-1,
2K in: 2*8.6 kJmol-1
= 37.9 kJ mol-1 needed, ~ one ATP
09/30/08 Biochemistry:Transport; Nucleic Acids
Diagram courtesy
Steve Cook
p. 7 of 50
What’s this
used for?


Sodium gets pumped back
in in symport with glucose,
driving uphill glucose
transport
That’s a separate passive
transport protein called
GluT1 to move glucose
back
09/30/08 Biochemistry:Transport; Nucleic Acids
Diagram courtesy
Steve Cook
p. 8 of 50
How do we transport big
molecules?

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Proteins and other big molecules often
internalized or secreted by endocytosis
or exocytosis
Special types of lipid vesicles created for
transport
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Receptor-mediated
endocytosis
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Bind macromolecule to specific receptor in
plasma membrane
Membrane invaginates, forming a vesicle
surrounding the bound molecules (still on the
outside)
Vesicle fuses with endosome and a lysozome
Inside the lysozyome, the foreign material
and the receptor get degraded
… or ligand or receptor or both get recycled
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Example: LDL-cholesterol
Diagram courtesy
Gwen Childs,
U.Arkansas for Medical
Sciences
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Exocytosis



Materials to be secreted are
enclosed in vesicles by the
Golgi apparatus
Vesicles fuse with plasma
membrane
Contents released into
extracellular space
Diagram courtesy
LinkPublishing.com
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Transducing signals

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Plasma membranes contain receptors
that allow the cell to respond to chemical
stimuli that can’t cross the membrane
Bacteria can detect chemicals:
if something useful comes along,
a signal is passed from the receptor to
the flagella, enabling the bacterium to
swim toward the source
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Multicellular
signaling

Hormones,
neurotransmitters, growth
factors all can travel to
target cells and produce
receptor signals
09/30/08 Biochemistry:Transport; Nucleic Acids
Diagram
courtesy
Science
Creative
Quarterly, U.
British
Columbia
p. 14 of 50
Extracellular
Signals



Internal behavior of
cells modulated by external influences
Extracellular signals are called first
messengers
7-helical transmembrane proteins with
characteristic receptor sites on
extracellular side are common, but
they’re not the only receptors
09/30/08 Biochemistry:Transport; Nucleic Acids
Image
courtesy
CSU
Channel
Islands
p. 15 of 50
Internal results of signals

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Intracellular: heterotrimeric G-proteins
are the transducers: they receive signal
from receptor, hydrolyze GTP, and emit
small molecules called second
messengers
Second messengers diffuse to target
organelle or portion of cytoplasm
Many signals, many receptors,
relatively few second messengers
Often there is amplification involved
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Roles of these systems
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Response to sensory stimuli
Response to hormones
Response to growth factors
Response to some neurotransmitters
Metabolite transport
Immune response
This stuff gets complicated, because the
kinds of signals are so varied!
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G proteins

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Transducers of external signals into the inside
of the cell
These are GTPases (GTP  GDP + Pi)
GTP-bound protein transduces signals
GDP-bound protein doesn’t
Heterotrimeric proteins; association of b and g
subunits with a subunit is disrupted by
complexation with hormone-receptor complex,
allowing departure of GDP & binding of GTP
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GTP
G protein
cycle

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Inactive
GDP
a
b
Ternary complex
g
disrupted by binding of
b
receptor complex
g
Ga-GTP interacts with
effector enzyme
GTP slowly hydrolyzed
away
Then Ga-GDP
reassociates with b,g
See fig. 9.39 for details
09/30/08 Biochemistry:Transport; Nucleic Acids
Active
a
GTP
H2O
Pi
a
GDP
Inactive
p. 19 of 50
Adenylyl cyclase


Cyclic
cAMP and cGMP:
AMP
second messengers
Adenylyl cyclase converts ATP to cAMP



Integral membrane enzyme; active site faces
cytosol
cAMP diffuses from membrane surface through
cytosol, activates protein kinase A
PKA phosphorylates ser,thr in target enzymes;
action is reversed by specific phosphatases
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O
N
N
Modulators
of cAMP
O
N
N
caffeine
O
N



Caffeine, theophylline inhibit cAMP
O
N
phosphodiesterase, prolonging cAMP’s
theophylline
stimulatory effects on protein kinase A
Hormones that bind to stimulatory receptors
activate adenylyl cyclase, raising cAMP levels
Hormones that bind to inhibitory receptors inhibit
adenylyl cyclase activity via receptor interaction
with the transducer Gi.
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H
N
N
O
O
Inositol-Phospholipid
Signaling Pathway
R1
R2
O
O
O

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2 Second messengers derived from O
phosphatidylinositol 4,5-bisphosphate
(PIP2)
Ligand binds to specific receptor;
signal transduced through G protein
called Gq
Active form activates
phosphoinositide-specific
phospholipase C bound to cytoplasmic
face of plasma membrane
09/30/08 Biochemistry:Transport; Nucleic Acids
P
O-
OH
O
OH
HO
O
HO
-O
P
O
p. 22 of 50
O
O
-O
PIP2
chemistry


Phospholipase C
hydrolyzes PIP2 to inositol
1,4,5-trisphosphate (IP3)
and diacylglycerol
Both of these products are
second messengers that
transmit the signal into the
cell
OP
O
P
O-
O
O-
O
OH
-O
OP
HO
O
O
OH
IP3
O
O
R1
R2
O
O
OH
diacylglycerol
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O
-O
OP
O
IP3 and calcium
P
O-
O
O
OOH
-O
OP
HO
O
O
OH



IP3 diffuses through cytosol and binds to a IP3
calcium channel in the membrane of the
endoplasmic reticulum
The calcium channel opens, releasing Ca2+ from
lumen of ER into cytosol
Ca2+ is a short-lived 2nd messenger too: it
activates Ca2+-dependent protein kinases that
catalyze phosphorylation of certain proteins
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O
O
R1
R2
O
O
Diacylglycerol and
protein kinase C



OH
diacylglycerol
Diacylglycerol stays @ plasma membrane
Protein kinase C (which exists in
equilibrium between soluble & peripheralmembrane form) moves to inner face of
membrane; it binds transiently and is
activated by diacylglycerol and Ca2+
Protein kinase C catalyzes
phosphorylation of a bunch of proteins
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Control of inositolphospholipid pathway


After GTP hydrolysis, Gq is inactive so I
no longer stimulates Phospholipase C
Activities of 2nd messengers are
transient


IP3 rapidly hydrolyzed to other things
Diacylglycerol is phosphorylated to form
phosphatidate
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Sphingolipids give rise to 2nd
messengers

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Some signals activate hydrolases that convert
sphingomyelin to sphingosine, sphingosine-1-P,
and ceramide
Sphingosine inhibits PKC
Ceramides activates a protein kinase and a
protein phosphatase
Sphingosine-1-P can activate Phospholipase D,
which catalyzes hydrolysis of
phosphatidylcholine; products are 2nd
messengers
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ligands
exterior
Receptor
tyrosine kinases


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Tyr kinase
monomers
interior
Most growth factors
function via a pathway
that involves these
enzymes
In absence of ligand, 2
nearby tyr kinase
molecules are separated
Upon substrate binding
they come together, form
a dimer
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Autophosphorylation
of the dimer


P
P
Enzyme catalyzes phosphorylation of
specific tyr residues in the kinase itself;
so this is autophosphorylation
Once it’s phosphorylated, it’s activate and
can phosphorylate various cytosolic
proteins, starting a cascade of events
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Insulin receptor



Insulin binds to an a2b2
tetramer;
binding brings b subunits
together
Each tyr kinase (b) subunit
phosphorylates the other one
The activated tetramer can
phosphorylate cytosolic
proteins involved in metabolite
regulation
09/30/08 Biochemistry:Transport; Nucleic Acids
Sketch courtesy of
Davidson College,
NC
p. 30 of 50
6
5
Pyrimidines

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N
4
1
2
N
3
Single-ring nucleic acid bases
pyrimidine
6-atom ring; always two nitrogens in the ring,
meta to one another
Based on pyrimidine, although pyrimidine itself
is not a biologically important molecule
Variations depend on oxygens and nitrogens
attached to ring carbons
Tautomerization possible
Note line of symmetry in pyrimidine structure
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H
N
O
Uracil and thymine


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
Uracil is a simple dioxo
derivative of pyrimidine:
2,4-dioxopyrimidine
Thymine is 5-methyluracil
Uracil is found in RNA;
Thymine is found in DNA
We can draw other
tautomers where we move
the protons to the oxygens
O
HN
uracil
HN
O
09/30/08 Biochemistry:Transport; Nucleic Acids
N
H
thymine
p. 32 of 50
O
Tautomers


Lactam and
Lactim forms
Getting these right
was essential to
Watson & Crick’s
development of
the DNA double
helical model
H
N
O
NH
O
HN
O
uracil - lactam
H
N
O
uracil - lactim
HN
HN
O
N
H
thymine - lactam
O
O
09/30/08 Biochemistry:Transport; Nucleic Acids
N
thymine - lactim
p. 33 of 50
OH
H
N
O
Cytosine
NH2
N
cytosine




This is 2-oxo,4-aminopyrimidine
It’s the other pyrimidine base found in
DNA & RNA
Spontaneous deamination (CU)
we’ll see the significance of that later
Again, other tautomers can be drawn
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Cytosine:
amino and imino forms

Again, this tautomerization needs to be
kept in mind
H
N
O
NH2
N
cytosine -amino form
H
N
O
NH
N
cytosine -imino form
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7
6
5
1N
Purines


8
4
2
N
N
3

H
N
9
Derivatives of purine; again, the purine
root molecule isn’t biologically
important
Six-membered ring looks a lot like
pyrimidine
Numbering works somewhat
differently: note that the glycosidic
bonds will be to N9, whereas it’s to
N1 in pyrimidines
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Adenine




This is 6-aminopurine
Found in RNA and DNA
We’ve seen how important adenosine
and its derivatives are in metabolism
Tautomerization happens here too
NH
NH2
H
N
N
N
N
adenine - amino form
H
N
HN
N
N
adenine - imino form
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Guanine
This is 2-amino-6-oxopurine
 Found in RNA, DNA
 Lactam, lactim forms
OH

O
H
N
H
N
N
HN
H2N
N
guanine - lactam
N
H2N
N
N
guanine - lactim
p. 38 of 50
09/30/08 Biochemistry:Transport; Nucleic Acids
Other natural purines


Hypoxanthine and xanthine
are biosynthetic precursors
of A & G
Urate is important in
nitrogen excretion
pathways
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Tautomerization and H-bonds


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Lactam forms predominate at neutral pH
This influences which bases are H-bond
donors or acceptors
Amino groups in C, A, G make H-bonds
So do ring nitrogens at 3 in pyrimidines
and 1 in purines
… and oxygens at 4 in U,T, 2 in C, 6 in G
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O
HO
Nucleosides
NR1R2
OH
HO
N-glycoside of ribofuranose



As mentioned in ch. 8, these are
glycosides of the nucleic acid bases
Sugar is always ribose or deoxyribose
Connected nitrogen is:


N1 for pyrimidines (on 6-membered ring)
N9 for purines (on 5-membered ring)
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Pyrimidine nucleosides

Drawn here in amino and lactam forms
OH
OH
HO
HO
OH
O
N
H2N
N
OH
O
cytidine
O
N
O
N
H
O
uridine
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OH
Pyrimidine
deoxynucleosides
OH
H
H
OH
O
N
O
N
H
OH
O
OH
2'-deoxyuridine
O
N
H
OH
N
O
2'-deoxythymidine
O
N
H2N
O
N
O
deoxycytidine
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A tricky nomenclature issue


Remember that thymidine and its
phosphorylated derivatives ordinarily
occur associated with deoxyribose, not
ribose
Therefore many people leave off the
deoxy- prefix in names of thymidine and
its derivatives: it’s usually assumed.
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Purine nucleosides

Drawn in amino and lactam forms
NH2
O
N
N
N
HN
N
N
H2N
N
N
O
O
HO
HO
OH
OH
HO
adenosine
HO
guanosine
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Purine deoxynucleosides
O
NH2
N
N
HN
N
N
H2N
N
N
N
O
O
OH
OH
HO
HO
deoxyadenosine
deoxyguanosine
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Conformations around the
glycosidic bond


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Rotation of the base around the glycosidic bond
is sterically hindered
In the syn conformation there would be some
interference between the base and the 2’hydroxyl of the sugar
Therefore pyrimidines are always anti, and
purines are usually anti
Furanose and base rings are roughly
perpendicular
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Glycosidic bonds

This illustrates the
roughly perpendicular
positionings of the
base and sugar rings
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Solubility of nucleosides and
lability of glycosidic linkages



The sugar makes nucleosides more
soluble than the free bases
Nucleosides are generally stable to basic
hydrolysis
Acid hydrolysis:


Purines: glycosidic bond fairly readily
hydrolized
Pyrimidines: resistant to acid hydrolysis
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Chirality in nucleic acids




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Bases themselves are achiral
Four asymmetric centers in
ribofuranose, counting the glycosidic
bond.
Three in deoxyribofuranose
Glycosidic bond is one of those 4 or 3.
Same for nucleotides:
phosphates don’t add asymmetries
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