Monomer, binds and stores O₂ in muscle. 

Single polypetide 152 amino acids long (16.7kDa).

78% of amino acids are found in the 8 α-helices.

O₂ transporter

Tetramer

2 α-globins(141aa)

2 β-globins (146aa)

Each subunit has a different amino acid sequence but very similar secondary and tertiary structure. Each subunit associates with one heme.

Globin found in leguminous plants

Sequester O₂ and protects

O₂-sensitive N₂- fixing bacteria.

8 α-helical segments (A-H) which fold together.

Connecting loops identified by the 2 adjacent positions (AB, EF)

Amino acids identifies by relative position.

F8

Directly bonded to Fe²⁺ 

E7

Close, but not bonded to heme.

O₂ binds to Fe²⁺ on E7 side of the atom

Blue

Hence oxygenated arterial blood is red.

Red

Hence venous blood

appears blue-ish.

HEME

CO binding vs. O₂ binding

CO binds free heme with 20 000x greater affinity than O₂

CO binds heme in Mb with only ~200x greater affinity than O₂

(due to steric hindrance and a H-bond between O₂ and HisE7)

Globions have a few amino acids which are identical though the coding genes and amino acid sequences are all very different

Secondary and tertiary structure well conserved.

• conformation of the ligand binding site
• molecular flexibility of the protein

1. hydrophobic interactions
2. hydrogen bonds
3. ion pairs (salt bridges)

• 30 occur at the interface of α₂-β₁ and that of α₁-β_2.
• 19 occur at the interface between α₂-β₁ and α₁-β_2.

ligand and modulator are identical

activator (+)or inhibitor (-)

i.e. for Hb O₂ is a ligand and an activating homotropic modulator 

ligand and modulator are different

can be

activators (+)

or

inhibitors (-)

primary: largely dissimilar (with       invariably key amino acids)

secondary: very similar

tertiary: veru similar

quaternary: very different (Mb         has no 4° structure)

 Physiological:

• H⁺ CO₂ (products of cellular respiration)
• BPG (intracellular metabolite)

Pathological

• CO (environmental toxin)

Hb has an affinity for CO 250x greaters than O₂

CO binding pushes Hb into the R state. Hb can still bind O₂ but cannot covert to the T state and release it. 

atm pO₂ = 20kPa ~ 250,000ppm

atm pCO (urban) = 4ppm

There is an inverse relationship between binding of O₂ and [H⁺]/[CO₂]

negative heterotropic allostery

HisH3: protonation fo HisHCs favours the formation of the ion-pair with AspFG1 and helps stabalize the transition state.

↓pH = Tstate = ↓O₂ binding

↑pH = higher O₂ affinity  

CO₂ binding at the amino terminal of Hb forms carbaminohemoglobin.

BPG binds to the central cavity of Hb and forms salt bridges with β subunits and stabalizes the quaternary structure of deoxy-Hb (T-state)

Important to the physiological adaptation to high altitude.

deoxy-Hb

low affinity

 low pH, high CO₂ and high 2,3 BPG favour T 

T favoured at low partial pressures

oxy-Hb

high affinity

high pH, low [CO₂],  low [2,3 BPG], and high partial pressure of oxygen favour the relaxed state. 

α₂γ₂

K_P for BPG is higher than adult Hb which gives HbF a higher affinity for oxygen.

M_r 540kDa

6 subunits:

→ 2 heavy chains (~220kDa each)

→ 4 light chains (~20kDa each)

Carboxy termini of the heavy chains are extended fibers that form a left-handed coiled-coil. Amino termini of each heavy chain has a large globular domain containing ATPase activity.

Light chains associate with globular proteins

In muscle cells, myosins form thick filaments.

M_r 42kDa

Globular monomers (G-actin) polymerize to form long filaments (F-actin).

Major protein of muscles.

In muscle thin filaments are made up of F-actin along with the protein tropomyosin and troponin.

1. allostery         (reversible, non-covalent)
2. reversible, covalent modification
3. interaction with regulatory proteins
4. proteolytic cleavage (covalent, non-reversible)

allosterically regulated enzyme

cAMP binds and produces a conformational change that releases the catalytic subunit.

(2cAMP bind to each regulatory subunit)

A regulatory enzyme with catalytic activity modulated by the non-covalent binding of a specific metabolite at a site other than the active site.

plot of V₀ vs. [S] is sigmoid

aspartate transcarbamoylase

(ATCase)

Allosteric enzyme that catalyses a commited step in biosynthesis of pyrimidine nucleotides (CMP, UMP).

Catalyzes formation of

N-carbamoylaspartate from aspartate and carbamoyl phosphate.

Binding of carbomoyl phosphate and aspartate by ATCase is cooperative.

N-(Phosphonacetly)-L-aspartate

(PALA)

T-state: closer conformation

(poor access for substrate)

R-state: open conformation

(better access for substrate)

Flexing at interfaces between c chains within the catalytic subunit. 

Catalytic trimers move apart (~12Å) and rotate relative to each other.

Regulatory dimers rotate ~15°.

Change in bend between R chains.

ATP (+)

disrupts ion pair → R state

CTP (-)

stabalized the ion pair → T state

ion pair between Asp236 on the catalytic chain and Lys143 on the regulatory chain connect the c and r subunits and stabalizes the T state.

*modulators bind to r chains whereas substrates bind at interfaces of c chains.

1. phophorylation 
2. adenylation
3. acetylation
4. ubiquitination
5. ADP-ribosylation

Ser

Lys

Tyr

His

Lys

amino terminus

Phosphorylation is sequence specfifc and occurs at Ser, Thr, Tyr, and His. 

Attachment of phosphoryl group is catalyzed by protein kinase

*Phosphoryl group can be removed by phosphoprotein phosphatase

Protein Kinase A

PKA

Serine/threonine kinase

concensus sqn: X-R-[RK]-X-[ST]-B

regulatory subunit: K-R-R-G-A-I

*cannot be phosphorylated

Cholera toxin ADP-ribosylates  G-proteins leading to the inactivation of GTPase activity.

Increased [cAMP] → activates PKA

In intestinal tract PKA activates CFTR  

(a Cl^- channel) and the Na⁺/H⁺ exhanger. This stimulates an efflux of NaCl

followed by H₂O leading to dehydration

and electrolyte loss.

relatively bulky group

charge 

O atoms (→ H-bonds)

site for protein-protein interactions

X-OH + ATP ↔ X-OPO₃^2-+ ADP + H⁺

Activates glycogen phosphorylase by phosphorylation.

Phosphorylation of Ser-14 disrupts the interaction between basic amino acids of N-terminus and acidic amino acids. Favours R state.

Scr-homology-2

(SH2)

Domains bind to sites containing phosphorylated to sites containing phosphorylated tyrosine (P-Tyr).

Autoinhibition and substrate-binfinh kinase Src is mediated by SH2 domain.

1. regulated expression of CDK and cyclin
2. subcellular localization of cyclins
3. binding of regulatory proteins (eg cyclin, p21)
4. phosphorylation of CDK

PSTAIRE helix moves into catalytic cleft (Glu51)

T loop changes conformation and position, allows access to catalytic cleft

Exposes Thr160 in T loop.

Phosphorylation of Thr160 in T loop.

Dephosphorylation of Tyr15 allows access for ATP

Phosphorylation of Thr160

T-loop forced out of catalytic cleft.

Thr160-PO₄ binds 3 Arg side-chains (including Arg50)

Stabalizes active conformation (R state)

P21

Cyclin-dependent kinase inhibitor 1

Regulatory protein that inhibits CDK.

Normally CDK2-cyclin E phosphorylates the retinoblastomas protein (pRb) which promotes cell division. If DNA is damaged p53 (transcription factor) induced expression of p21 which binds CDK2 activity which blocks CDK2 activity. (pRb not phosphorylated and cell division stops. p53 mutated in agressive cancers.

• external/internal signals
• cell surface receptors
• "initiator" caspases
• mitchondria → cytochrome C
• effector caspases 

*caspases are synthesized as proenzymes and activated by proteolysis

Digestive enzyme which is useful for breaking down food but must be controlled. 

Serine proteases.

Highly sensitive to pH.

His57 must not be protonated.

Ile16 must be protonated. 

Catalyzes step one of glycolysis

↑ [glucose] (+) hexokinase

↑ [F6-P] (-) hexokinase 

phosphofructokinase-1

PFK-1

Catalyses step 3 of glycolysis.

Commits fructose 6-phosphate to glycolysis.

↑ ATP (-) PFK-1

↑ AMP,ADP (+)PFK-1

↑ citrate (-)PFK-1

↑ F2,6-BP (+) PFK-1

Pyruvate Kinase

PK

catalyzes step 10 of glycolysis

↑ ATP  (-) PK

↑ acetyl-CoA (-)PK

↑ long chain FA (-) PK

↑ alanine (-) PK

↑ F1,6-BP (+) PK

isoenzyme in muscle

Normally has max activity, allosterically inhibited by G6-P

↑ activity at ↓ [glucose]

Hexokinase IV 

glucokinase

expressed 

↓ affinity for glucose

inhibited IV is

inhibited by F6-P

Inhibition effected through glucokinases regulatory protein

Helps catalyze conversion of pyruvate to phosphoenolpyruvate during gluconeogenesis

(mitochondria)

acetyl-CoA (+)

ADP (-)

phosphoenolpyruvate carboxykinase

(PEP carboxykinase)

catalyzes last step of pyruvate to phosphoenolpyruvate.

ADP (-)

*One advantage to the mitochondrial bypass to PEP in gluconeogenesis is it provides NADH in the cytosol.

Conversion of F1,6-BP to F6-P

Step 8 of gluconeogenesis

bypass for step 3 of glycolysis

ATP (-)

ADP (+)

AMP (+)

citrate (-)

F26BP (+)

phosphofructokinase-2 (PFK-2)

fructose 2,6-bisphosphatases (FBPase2)

PFK-2/FBPase-2 is a bifunctional enzyme regulated by hormone controlled phosphorylation and xylulose 5-phosphate

step 10 of gluconeogenesis

(bypass for step1 of glycolysis)

Glucose 6-phosphate + H₂O → glucose + Pi

Liver isoenzyme

(phosphorylation of

PFK-2/FBPase-2)

Cardiac isoenzyme

(regulation of

PFK-2/FPBase-2)

Oxidative

oxidation of G6-P 

Non-oxidative

isomerization rearrangments

back to glucose 6-phosphate?

transketolase

tranldolase

in pentose phosphate pathway:

activates phosphatase that stimulates liver PFK-2

regulated dephosphorylation of PFK-2/FBP

Regulation of 

pentose phosphate pathway

1. Glucose 6-phosphate dehydrogenase 

NADP⁺  (+)

NADPH  (-)

2. reactions of non-oxidative phases are driven by [substrate]

polymer of glucose for storage

quick source of energy

metabolised in by liver by glycolysis and control of blood [glucose]

metabolised in the muscle by glycolysis

Primes glycogen synthesis

• glucosyltransferase
• glucose is tranferred from UDP-glucose to the glycogenin protein
• additional glucose units are added to a total of 8
• glycogen synthase can then take over

involved in glycogen synthesis

glucose 6-P → glucose 1-P

Involved in glycogen synthesis

glucose 1-P + UTP → UDP-glucose + PPi  

Glycogen synthase kinase 3

(GSK-3)

Phosphorylation of glycogen synthase by casein kinase allows GSK-3 to bind glycogen synthase, phosphorylate it, and inactivate it.

When GSK-3 is phosphorylated it is inactivated.

enzyme that controls glycogen phosphorylase

phophorylates glycogen phosphorylase → this activates

glycogen phosphorylases b kinase is activated by PKA-mediated phosphorylation

• epi or glucagon trigger G-protein ↑[cAMP]
• ↑ PKA activity 
• PKA phosphorylates glycogen phosphorylase b kinase
• GPbK in turn phosphorylates glycogen phosphorylase
• ↑ glycogenolysis 
• dephosphorylation by glycogen phosphorylase a kinase is favoured by allosteric effect of glucose binding

↑[glucose]_blood → release of insulin

↓ glucose breakdown

↑ glycogen synthesis

↑ glycolysis

↓[glucose]_blood → release of glucagon

↑ glucose breakdown

↓ glycogen synthesis

↓ glyoclysis

(↑ gluconeogenesis in the liver)

Causes the liver to import and use/store glucose

fed state

causes the liver to mobilize glucose for export to blood

PKA plays a central role in this response

fasted state

acts on liver!

prepares the body for activity

acts on liver and muscle

↑ glycogenolysis

↑ gluconeogenesis

export of glucose in the liver

fuel consumption by muscle cells

(↑ glycolysis/glycogenolysis in muscle)

*glycolysis does not increase in liver

initiator caspase

...become activated through proteolytic cleavage in response to an apoptotic signal. Active caspase 8 then proteolitically cleaves intracellular targets, including mitochondria (causing release of cytochrome C), and leads to the activation of effector caspases.

effector caspase

lead to destruction of cellular proteins

Asp-His-Ser

sensitive to pH

[O₂] at which half of the available ligand-binding sites are occupied

p50