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6 March 2008
Membrane Signaling;General Metabolism I
Andy HowardIntroductory Biochemistry
6 March 2008
6 March 2008Signaling; General Metabolism I Page 2 of 40
What we’ll discuss Membrane Signaling
General Principles G proteins Adenylyl cyclase Inositol-phospholipid
signaling pathway Receptor tyr kinases
Metabolism Definitions Pathways Control Feedback Phosphorylation Thermodynamics Kinetics
6 March 2008Signaling; General Metabolism I Page 3 of 40
Extracellular Signals
Internal behavior ofcells 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
Image courtesy CSU Channel Islands
6 March 2008Signaling; General Metabolism I Page 4 of 40
Internal results of signals
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
6 March 2008Signaling; General Metabolism I Page 5 of 40
Roles of these systems 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!
6 March 2008Signaling; General Metabolism I Page 6 of 40
G proteins 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 and
subunits with subunit is disrupted by complexation with hormone-receptor complex, allowing departure of GDP & binding of GTP
6 March 2008Signaling; General Metabolism I Page 7 of 40
G protein cycle
Ternary complex disrupted by binding of receptor complex
G-GTP interacts with effector enzyme
GTP slowly hydrolyzed away
Then G-GDP reassociates with ,
See fig. 9.39 for details
GDP
GTP
GTP
Inactive
Active
GDP
H2O
Pi
Inactive
6 March 2008Signaling; General Metabolism I Page 8 of 40
Adenylyl cyclase
cAMP and cGMP: 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
Cyclic AMP
6 March 2008Signaling; General Metabolism I Page 9 of 40
Modulators of cAMP
Caffeine, theophylline inhibit cAMP phosphodiesterase, prolonging cAMP’s 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.
O N
N
N
N
O
caffeine
HN
NNO
N
O
theophylline
6 March 2008Signaling; General Metabolism I Page 10 of 40
Inositol-Phospholipid Signaling Pathway 2 Second messengers derived from
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
O
HO
HO
O
OH
OHPO O-
O
O
O
R1
O
O R2
P
O
O-O
6 March 2008Signaling; General Metabolism I Page 11 of 40
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
O
OH
HO
O
O
OH
P
O
-OO-
IP3
P O-O
-O
P
O-
OO-
OH
O
O
R1
O
O R2
diacylglycerol
6 March 2008Signaling; General Metabolism I Page 12 of 40
IP3 and calcium
IP3 diffuses through cytosol and binds to a 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
O
OH
HO
O
O
OH
P
O
-OO-
IP3
P O-O
-O
P
O-
OO-
6 March 2008Signaling; General Metabolism I Page 13 of 40
Diacylglycerol and protein kinase C
Diacylglycerol stays @ plasma membrane
Protein kinase C (which exists in equilibrium between soluble & peripheral-membrane 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
OH
O
O
R1
O
O R2
diacylglycerol
Protein kinase C(PDB 1APM)
6 March 2008Signaling; General Metabolism I Page 14 of 40
Control of inositol-phospholipid 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
6 March 2008Signaling; General Metabolism I Page 15 of 40
Sphingolipids give rise to 2nd messengers 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 PlaseD, which
catalyzes hydrolysis of phosphatidylcholine; products are 2nd messengers
6 March 2008Signaling; General Metabolism I Page 16 of 40
Receptor tyrosine kinases
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
exterior
interior
ligands
Tyr kinase monomers
6 March 2008Signaling; General Metabolism I Page 17 of 40
Autophosphorylation of the dimer
Enzyme catalyzes phosphorylation of specific tyr residues in the kinase itself; so this is autophosphorylation
Once it’s phosphorylated, it’s activated and can phosphorylate various cytosolic proteins, starting a cascade of events
PP
6 March 2008Signaling; General Metabolism I Page 18 of 40
Insulin receptor Insulin binds to an 22
tetramer;binding brings subunits together
Each tyr kinase () subunit phosphorylates the other one
The activated tetramer can phosphorylate cytosolic proteins involved in metabolite regulation
Sketch courtesy ofDavidson College, NC
6 March 2008Signaling; General Metabolism I Page 19 of 40
Metabolism Almost ready to start the specifics
(chapter 11) Define it!
Metabolism is the network of chemical reactions that occur in biological systems, including the ways in which they are controlled.
So it covers most of what we do here!
6 March 2008Signaling; General Metabolism I Page 20 of 40
Intermediary Metabolism Metabolism involving small molecules Describing it this way is a matter of
perspective:Do the small molecules exist to give the proteins something to do, or do the proteins exist to get the metabolites interconverted?
6 March 2008Signaling; General Metabolism I Page 21 of 40
Anabolism and catabolism Anabolism: synthesis of complex
molecules from simpler ones Generally energy-requiring Involved in making small molecules and
macromolecules Catabolism:degradation of large
molecules into simpler ones Generally energy-yielding All the sources had to come from
somewhere
6 March 2008Signaling; General Metabolism I Page 22 of 40
Common metabolic themes Maintenance of internal concentrations
of ions, metabolites, enzymes Extraction of energy from external
sources Pathways specified genetically Organisms & cells interact with their
environment Constant degradation & synthesis of
metabolites and macromolecules to produce steady state
6 March 2008Signaling; General Metabolism I Page 23 of 40
Metabolism and energy
6 March 2008Signaling; General Metabolism I Page 24 of 40
Pathway A sequence of reactions such that
the product of one is the substrate for the next
Similar to an organic synthesis scheme(but with better yields!)
May be: Unbranched Branched Circular
6 March 2008Signaling; General Metabolism I Page 25 of 40
Why multistep pathways?
Limited reaction specificity of enzymes
Control of energy input and output: Break big inputs into ATP-sized inputs Break energy output into pieces that
can be readily used elsewhere
6 March 2008Signaling; General Metabolism I Page 26 of 40
Regulation Organisms respond to change
Fastest: small ions move in msec Metabolites: 0.1-5 sec Enzymes: minutes to days
Flow of metabolites is flux: steady state is like a leaky bucket Addition of new material replaces the
material that leaks out the bottom
6 March 2008Signaling; General Metabolism I Page 27 of 40
Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki
6 March 2008Signaling; General Metabolism I Page 28 of 40
Feedback and Feed-forward
Mechanisms by which the concentration of a metabolite that is involved in one reaction influences the rate of some other reaction in the same pathway
6 March 2008Signaling; General Metabolism I Page 29 of 40
Feedback realities Control usually exerted at first
committed step (i.e., the first reaction that is unique to the pathway)
Controlling element is usually the last element in the path
6 March 2008Signaling; General Metabolism I Page 30 of 40
Feed-forward
Early metabolite activates a reaction farther down the pathway
Has the potential for instabilities, just as in electrical feed-forward
Usually modulated by feedback
6 March 2008Signaling; General Metabolism I Page 31 of 40
Activation and inactivation by post-translational modification
Most common:covalent phosphorylation of protein
usually S, T, Y, sometimes H Kinases add phosphate
Protein-OH + ATP Protein-O-P + ADP… ATP is source of energy and Pi
Phosphatases hydrolyze phosphoester:Protein-O-P +H2O Protein-OH + Pi
… no external energy source required
6 March 2008Signaling; General Metabolism I Page 32 of 40
Phosphorylation’s effects
Phosphorylation of an enzyme can either activate it or deactivate it
Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated
Example:glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme
6 March 2008Signaling; General Metabolism I Page 33 of 40
Glycogen phosphorylase Reaction: extracts 1 glucose
unit from non-reducing end of glycogen & phosphorylates it:(glycogen)n + Pi (glycogen)n-1 + glucose-1-P
Activated by phosphorylationvia phosphorylase kinase
Deactivated by dephosphorylation byphosphorylase phosphatase
6 March 2008Signaling; General Metabolism I Page 34 of 40
Amplification
Activation of a single molecule of a protein kinase can enable the activation (or inactivation) of many molecules per sec of target proteins
Thus a single activation event at the kinase level can trigger many events at the target level
6 March 2008Signaling; General Metabolism I Page 35 of 40
Evolution of Pathways:How have new pathways evolved? Add a step to an existing pathway Evolve a branch on an existing pathway Backward evolution Duplication of existing pathway to create
related reactions Reversing an entire pathway
6 March 2008Signaling; General Metabolism I Page 36 of 40
Adding a step
A B C D E P
• When the organism makes lots of E, there’s good reason to evolve an enzyme E5 to make P from E.
• This is how asn and gln pathways (from asp & glu) work
E1 E2 E3 E4 E5
Original pathway
6 March 2008Signaling; General Metabolism I Page 37 of 40
Evolving a branch Original pathway:
D A B C X
Fully evolved pathway: D A B C X
E1 E2E3
E3a
E3b
6 March 2008Signaling; General Metabolism I Page 38 of 40
Backward evolution
Original system has lots of E P E gets depleted over time;
need to make it from D, so we evolve enzyme E4 to do that.
Then D gets depleted; need to make it from C, so we evolve E3 to do that
And so on
6 March 2008Signaling; General Metabolism I Page 39 of 40
Duplicated pathways
Homologous enzymes catalyze related reactions;this is how trp and his biosynthesis enzymes seem to have evolved
Variant: recruit some enzymes from another pathway without duplicating the whole thing (example: ubiquitination)
6 March 2008Signaling; General Metabolism I Page 40 of 40
Reversing a pathway We’d like to think that lots of pathways are fully
reversible Usually at least one step in any pathway is
irreversible (Go’ < -15 kJ mol-1) Say CD is irreversible so E3 only works in the
forward direction Then D + ATP C + ADP + Pi allows us to
reverse that one step with help The other steps can be in common This is how glycolysis evolved from
gluconeogenesis