6 March 2008 Membrane Signaling; General Metabolism I Andy Howard Introductory Biochemistry 6 March 2008

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


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


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


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!


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


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


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


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


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


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


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-


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)


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


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


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


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


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


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!


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?


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


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


Metabolism and energy


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


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


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


Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki


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


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


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


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


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


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


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


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


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


Evolving a branch Original pathway:

D A B C X

Fully evolved pathway: D A B C X

E1 E2E3

E3a

E3b


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


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)


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

Documents

6 March 2008 Membrane Signaling; General Metabolism I Andy Howard Introductory Biochemistry 6 March 2008