Protein Function/Enzyme Regulation/Biosignalling

Chpts. 5, 6, 12

1o, 2o, 3o, 4o StructuresREMEMBER??

• Structure defines function

• If >1 polypeptide chain, 4o structure– Chains not independent

• Book: Proteins are dynamic structures

Definitions (Chpt. 5)

• Ligand – bound reversibly to prot

• Binding site – where ligand binds – Complementarity

• Induced fit – protein flexes greater complementarity

• (What do all of these characteristics remind you of?)

Protein/Ligand Binding

• May be regulated by another molecule

– May be second ligand w/ second binding site

• Second ligand interaction flexing change in ability of first ligand to bind

– First ligand may bind better or worse under influence of second ligand

Hemoglobin (Hb)

• 4 polypeptide chains + 4 heme grps

• MW 64,5000

old book

Protein (Globin)

• Globular

• 4 hydrophobic pockets

• 4o structure due to interaction ~30 aa’s

/ subunits interact (not / or /)

– Mostly hydrophobic interactions, some ionic

• Heme

– Protophorphyrin ring

– Binds single Fe as Fe+2

– Sim to pigments

– Resonance (electron transport, color, UV absorbance)

Fe Held in Heme; Heme Held in Hb

• 6 Coordination sites for heme Fe

– 4 Bind N’s of protoporphyrin ring

– 1 Binds globin His R grp N

– 1 Binds O2

Changes in heme electronic prop’s

Color change

•Fe can also bind CO

• Globin may be in T state or R state

– T state more stable w/out O2

– O2 prefers to bind globin in R state (either poss.)

– Bound O2 stabilizes R

O2 Binding to Heme Influences Globin• T state (stable deoxyHb) binds O2

Shift in globin conform’n , subunits slide, rotate

subunits closer

• R state results

• O2 binding @ Fe incr’d planarity of heme altered interactions of R grps of nearby aa’s

Imptc to Hb Function (Transport O2)

• O2 must be reversibly bound to Hb, but tight enough for transport

• Binding of 1 O2 molecule causes TR

– R state now stabilized

– Subunits have been effected

• Now easier for 2nd O2 to bind

• 3rd, 4th O2’s

– R state strengthened w/ each O2 added

Allosteric Protein

• Ligand binding @ one site affects binding abilities @ other sites on same protein

• Due to conform’l changes altered binding site(s)

• Hemoglobin example

– O2 = activator (stimulator)

– Positive cooperativity among subunits

• Can be treated mathematically

– Similar to Ka, Kd

– P + nL PLn

• P = Protein

• L = ligand

= binding sites occ’d/total binding sites

• Based on fraction of binding sites occupied, derive Hill coefficient (hH)

– = 1, no cooperative binding

– < 1, negative cooperativity

– 1, positive cooperativity

Models for Cooperativity

• Monod

– “All or nothing”

– No subunit in any independent conformation

– Ligand binds any, but prefers one

• Koshland (Sequential)

– Subunits more independent

– One subunit acts as modulator

•Conform’l change influences conform’l changes in other subunits

– “Graded effects”

Sickle Cell Anemia

• Mutation single improper aa in Hb globin

chain Glu Val

– Now – charge uncharged side chain

“sticky” hydrophobic pt @ outer Hb surface

• DeoxyHb mol’s associate w/ each other

Strand, fiber form’n

Long, thin crescent rbc’s

Allosteric Effects Regulate Some Enz Activity in Metabolism

• Metab pathways mediated by enzymes

– Several rxns in succession

– Each rxn catalyzed by partic enzyme

– P rxn 1 becomes S for rxn 2, etc.

Enzymes Can Be Inhibited

• Product inhib’n

– Enz may be inhib’d by its own P

– Inverse relationship of [P] and further P synthesis

– P acts as competitive inhibitor

•Resembles S

•Fits enz active site

•Competes

• Inhib’n overcome

• Feedback inhibition– Enz may be inhib’d by metabolite

from further down pathway

– L-ileu prevents form’n

– Inhibits thr dehydratase

•No other

– Thr dehydratase = regulatory enzyme

•Regulates pathway

Regulatory Enzymes

• Catalyze slowest step

• Stim’d or inhibited

• Commonly 1st

• Point of commitment

• May be allosteric OR controlled by covalent modification

Allosteric Regulatory Enzymes

• REMEMBER how Hb worked

• Modulated

• >1 binding site

– Binding of S to one site affects other binding site(s)

– Both need not be catalytic

• Often 1 regulatory

– Both specific for S or modulator

– Often on diff subunits

• Modulator binding at regulatory site conform’l change at catalytic site

– May be harder or easier for S to bind

– Conform’l changes due to noncovalent interactions

Altered Kinetics of Allosteric Enzymes

• M-M model hyperbolic

• Allosteric model sigmoidal

– If modulator stimulates, more hyperbolic

– If modulator inhibits, more sigmoidal

• KM changes

Covalently Modified Regulatory Enzymes

• Also controlled through modulators

• Now modulator covalently bound

– At some funct’l grp of aa of enz 1o structure

– Need OTHER enz’s to catalyze binding of modulator

– Need EVEN OTHER enz’s to catalyze lysis of modulator

– So have groups of enzymes

• Not necessarily subunits that interact

Covalent Modification at Reg Enz Funct’l Grps

• Could disrupt entire 2o, 3o structure

• Could inhibit S approach

• Could inhibit S fit

• Could modify funct’l grps impt to catalysis

Types of Modification• Phosphorylation Nucleotidation

• ADP-ribosylation Methylation/acetylation

Glycogen Phosphorylase Example• Glycolysis reg enz

• 2 subunits, each w/ ser (what’s it’s funct’l grp?)

• Cleaves glycogen (what’s it made of?)

– Releases glu then phosphorylates glu

• 2 forms of enz (a = active, b = inactive)

• 2 associated enz’s

– Phosphorylase kinase cat’s b a

• Active form – phosphorylated

• W/ transfer of Pi from ATP

– Phosphorylase phosphatase cat’s a b

• W/ hydrolysis Pi

• Phosph’n interferes w/ stabilizing ionic interactions

– Changes folding (what type of structure (1o, etc.) is most impt to folding?)

– New interactions between diff aa’s

– Incr’s catalytic activity

• a, b forms differ in 2o, 3o, 4o structures also

– So some allosteric properties

Another Kinase May Activate Glycogen Phosphorylase

• Protein kinase A is modulated also

• Works through 2nd messenger system:

Phosphorylation & Second Messenger Systems• Involves both allosteric &

cov’ly mod’d enzymes

• “First messenger” = non-lipid hormone

– Binds cell membr receptor

– Book ex: epinephrine

Conform’l changes in membr-bound proteins

– Receptor, G-proteins, adenylyl cyclase

– All are allosteric prot’s

Act’n adenylyl cyclase

– Cat’s rxn ATP cAMP

• cAMP (what type of molecule?)

– “Second signal”

– Regulates activities of many enzymes

– Act’s (usually) regulatory enz’s through allosteric control

• Protein kinase A

– 4 subunits (2 regulatory, 2 catalytic)

– cAMP binding of cAMP to regulatory units

Allosteric change

Catalytic subunits dissociate

Catalytic subunits activated

• For glycogen phosphorylase example:

– PKA subunit dissociation

Activation of PKA

Phosph’n glycogen phosphorylase b

Activation glycogen phosphorylase (now “a” form)

• “Signal transduction cascade” = amplification of signal

• PKA regulates many enzymes

• cAMP regulates many pathways

– Metabolic, others

• Caffeine (methylated purine) inhibits breakdown of cAMP

– What type of inhibitor might it be?

– What pharmacologic effects would you expect from caffeine?