09/30/08biochemistry:transport; nucleic acids transport & signaling; nucleic acid chemistry andy...

09/30/08Biochemistry:Transport; Nucleic Acids

Transport & Signaling;Nucleic Acid Chemistry

Andy HowardIntroductory Biochemistry

30 September 2008

09/30/08 Biochemistry:Transport; Nucleic Acids p. 2 of 50

What we’ll discuss Membrane transport

Energetics Active transport Transporting big

molecules

Membrane Signaling Adenylyl cyclase Inositol-phospholipid

signaling pathway Receptor tyr kinases

Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G

Nucleosides


Protein-facilitated passive transport

All involve negative Gtransport

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

Diagram courtesySaint-Boniface U.


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.


Primary active transport

Energy source:usually ATP or light

Energy source directlycontributes to overcomingconcentration gradient 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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

BacteriorhodopsinPDB 1F50, 1.7Å25 kDa monomer


Secondary active transport

Active transport of one solute is coupled to passive transport of another

Net energetics is (just barely) favorable

Generally involves antiport Bacterial lactose influx driven by

proton efflux Sodium gradient often used in

animals

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Pyrococcus Multi-sugar transporterPDB 1VCI83 kDa dimer


Complex case: Na+/K+ pump

Typically [Kin] = 140mM, [Kout] = 5mM,[Nain] = 10 mM, [Naout] = 145mM.

ATP-driven transporter:3 Na+ out for 2 K+ inper 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

Diagram courtesy

Steve Cook


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

Diagram courtesy

Steve Cook


How do we transport big molecules? Proteins and other big molecules often

internalized or secreted by endocytosis or exocytosis

Special types of lipid vesicles created for transport


Receptor-mediated endocytosis

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


Example: LDL-cholesterol

Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences


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


Transducing signals 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


Multicellular signaling

Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals

Diagram courtesy Science Creative Quarterly, U. British Columbia


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


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 Phospholipase D,

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 activate 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


Pyrimidines Single-ring nucleic acid bases 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

N

N

pyrimidine

1

2

3

4

5

6


Uracil and thymine 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

HN

OHN O

uracil

HN

O NH

O

thymine


Tautomers

Lactam and Lactim forms

Getting these right was essential to Watson & Crick’s development of the DNA double helical model

HN

OHN O

uracil - lactam

NH

ONO

uracil - lactimH

HN

O NH

O

thymine - lactam

HN

O N OH

thymine - lactim


Cytosine

This is 2-oxo,4-aminopyrimidine It’s the other pyrimidine base found in

DNA & RNA Spontaneous deamination (CU)

we’ll see the significance of that later Again, other tautomers can be drawn

N

OHN NH2

cytosine


Cytosine:amino and imino forms Again, this tautomerization needs to be

kept in mind

N

OHN NH

cytosine -imino form

N

OHN NH2

cytosine -amino form


Purines Derivatives of purine; again, the

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

HN

NN

N

purine

1

2

3

4

56 7

8

9


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

N

N

NH2

N

HN

adenine - amino form

HN

N

NH

N

HN

adenine - imino form


Guanine This is 2-amino-6-oxopurine Found in RNA, DNA Lactam, lactim forms

HN

NNH2N

HN

O

guanine - lactam

HN

NNH2N

N

OH

guanine - lactim


Other natural purines Hypoxanthine and xanthine

are biosynthetic precursors of A & G

Urate is important in nitrogen excretion pathways


Tautomerization and H-bonds 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


Nucleosides

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)

NR1R2

OH

HO

O

HO

N-glycoside of ribofuranose


Pyrimidine nucleosides Drawn here in amino and lactam forms

OH

OHHO

ON

ONH2N

cytidine

OH

OHHO

ON

ONH

O

uridine


Pyrimidine deoxynucleosides

OH

OHH

ON

ONH

O

2'-deoxyuridine

OH

OHH

ON

ONH

O

2'-deoxythymidineOH

OH

ON

ONH2N

deoxycytidine


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.


Purine nucleosides

Drawn in amino and lactam forms

OH

HO

HO

O

N

N

NH2

N

N

adenosine

OH

HO

HO

O

N

N

O

HN

H2N N

guanosine


Purine deoxynucleosides

OH

HO

O

N

N

O

HN

H2N N

deoxyguanosine

OH

HO

O

N

N

NH2

N

N

deoxyadenosine


Conformations around the glycosidic bond 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


Glycosidic bonds

This illustrates the roughly perpendicular positionings of the base and sugar rings


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


Chirality in nucleic acids 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

09/30/08biochemistry:transport; nucleic acids transport & signaling; nucleic acid chemistry andy...

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