What is a globular protein?

Give examples?

• A globular protein is different segments of a polypeptide chain that fold back on each other
• Egs. include enzymes, Igs, transport proteins & regulatory proteins

Describe 1°, 2°, 3° & 4° structures of proteins.

What shape do non-covalent bonds permit?

What's important to know about 4° structure

• 1° - linear strand of aas held together via peptide bonds
• 2° - fold back on each other, held together by H⁺ bonds, also see α-helices & β-pleated sheets
• 3° - disulfide bridges, wound so center is hydrophobic
• 4° - simple polypeptides coming together, non-covalent bonds

• non-covalent bonds preserve overall molecular integrity while permitting meta-stable shape, underlies inter- and intramolecular comm & flexibility, esp in intracellular space

• Hb is 4° structure

What is an essential amino acid?

How many are there?

Name them

• The body does not synthesize them so they must be gained from the diet
• 8 (sometimes 10)
• M, I, L, K, F, T, W, V (H is also considered essential)

Name the following

Hydrophobic aas

Hydrophilic aas

Acidic aas

Basic aas

aas that acquire an -OH group

• non-polar (G, A, P, V, L, I, M)
• polar - (S, T, N, Q)
• proton donors (D, E)
• proton acceptors (K, R, H)
• S, T, Y

How are non-essential aas synthesized?

They are synthesized using glycolytic pathways & phenylalanine

Heme + globin = _______?

Heme is a ______ group of hemoglobin

Which globular protein is monomeric? multimeric?

• holoenzyme

• prosthetic

• myoglobin, hemoglobin

How many Hb mlcs to one RBC?

Does the RBC have organelles?

What's special about its shape?

• 300 million

• No - no nuclei, mitochondria, protein synthesis

• optimal volume for max amnt of surface area exposed

Which state does Iron need to be in for Hb to be active?

Where does it form a bond?

What forms a bond on the other end?

What happens with every breakdown of heme?

• Iron must be in the ferrous (Fe⁺²) state to be active

• Fe⁺² forms a bond with proximal histidine 

• During oxygenation O₂ forms a bond on the distal histidine side

• Every mlc of heme broken down yields CO (& Fe⁺³) (out competes O₂)

What does Hb transport look like in the lungs?

What happens to Hb on way back to lungs from tissue?

What happens in the muscle?

• HbO₂, there is no chemical bond, reversible (de)-oxygenation

• some CO₂ is covalently bound to terminal -NH₂ groups in negatively charged Carbamino group (20%)

• In muscle, MbO₂ (it's the reserve tank)

Which has a higher affinity for O₂, Mb or Hb?

How many globins does Hb have? Mb?

• Mb has a much higher affinity for pO₂

• 4, 1 (but both have identical helices)

What color is oxygenated blood? deoxygenated blood? carboxy Hb?

____ is O₂ deficiency, while ____ is an absence of O₂.

To what condition can the former (above) lead?

• red, blue, bright cherry-red

• hypoxia, anoxia

• cyanosis

What kind of curve is Hb?

What is the avg PO₂ of deoxygenated blood returning to the heart? PO₂ of blood leaving the lungs?

What % of the O₂ in arterial blood is released to tissues during rest or light exercise?

What % is held by Hb & can be released to tissues w/ low PO₂?

• Sigmoidal, saturation curve

• 40mmHg, 100mmHG

• 25%

• 75%

T or F, Mb undergoes a conformational change?

Where is Mb located?

What is the structure of Mb?

Over 80% of the aa residues of Mb participate in formation of ______ α-helices

• False, it only binds on O₂

• in the muscle only

• single polypeptide chain

• amphipathic

Are disulfide bridges present in either Hb or Mb? what's the consequence?

No, neither. Makes Hb and Mb unstable structures

What type of curve is Mb?

What is P₅₀? for Mb? Hb?

What drives the (un) loading of O₂?

What's the relationship btw affinity & P₅₀?

Shifting in what direction increases affinity?

• steep, hyperbolic curve (Michaelis-Menten)

• point of 1/2 O₂ saturation, 1, 26

• pO₂

• inverse

• left

What is the pH of HbA?

Does HbF have a higher or lower affinity for O₂?

What about a llama?

• 7.4

• higher (curve shifts to the left)

• haha, higher than HbF in case you were curious

What makes up the tetramer of HbA?

What accounts for differences in Hb?

What are 3 types of Hb modifications?

• 2 non-identical pairs of α-globin & β-globin chains (dimers are identical)

• globin variants

• HbS - switch glu → val on β chain (note: val is hydrophobic aa so it shrinks & pulls in towards itself)
• HbC - switch glu → lys on β chain (note: in homozyg mild chronic hemolysis, no specific therapy)
• HbA_1c - glycated Hb, see in DM

Chr 16 has __ genes for __ chain(s) and is present in the embryo.

Which 2 other types of globin chains are present in the embryo?

Chr 11 has __ genes for __ chain(s) and is present in HbA only (not HbF), consequences?

Which one is present in fetal stages?
Which one found in minor adult HbA2?

• 2, α-globin

• ζ (α-like) & ε (β-like) (1 gene each)

• 1, β-globin, increase in deficiencies

• 1 δ

• 2 γ in HbF

What does the oxidation of Fe⁺² to Fe⁺³ produce?

What can cause excess production of this?

• Creates a non-functional Methemoglobin or Metmyoglobin, a useless O₂ transporter, reduces O₂ affinity

• oxidizing chemical such as tobacco smoke, inorganic nitrites, aniline dyes

Name 3 reducing substances that help the RBC defend against oxidation of its heme iron, which is 1°?

What does proper binding of heme to globin create?

Dissociation of heme from globin permits what?

• Ascorbate (Vit C) & glutathione (made up of Glu, Gly, Cys), NADH (from glycolysis) is the 1° one

• a protective environment for Fe⁺²

• Oxidation to hemin (Fe⁺³)

What's a common cause of congenital methemoglobinemia (HbM)?

How do you tx?

• Substitution of proximal histidine by tyrosine

• Methylene blue

What reduces methemoglobin to normal Hb?

What's the equation?

• Methemoglobin reductase

• Hemin (Fe⁺³) + NADH + H⁺ → Heme (Fe⁺²) + NAD⁺

What happens to the Hb curve when CO binds?

What happens to O₂?

Man's tolerance for CO is low at 1% why?

• Sat'n curve shifts left

• O₂ can't be released

• It outcompetes O₂ so it can't be very high

What 2 major Hb functions does CO affect?

• Impairs the carrying capacity of O₂
• Impairs the carrying capacity of CO₂

What are the two states of Hb?

What causes the states to change?

Which side does O₂ bind first?

• Unoxygenated (Tense state) - hydrophobic, ionic & H⁺ bonds, tighter Hb
• Oxygenated (Relaxed state) - some H⁺ & salt bonds broken, looser Hb

• oxygen, causes cooperative binding

• O₂ binds to the proximal histidine, distal histidine controls access to binding (note: a marker, Val FG5 is pushed

T or F changes in pO₂ far from the normal range have maximal effect on HbO₂ associaton. If F, make it true.

False, minimal effect

What does "positive cooperativity" mean and why is it important?

• Positive cooperativity means that as heme #1 is oxygenated, Hb shifts toward R state with a higher O₂ affinity, repeated for heme #2, 3 & 4
• It's impt because it decreases the fold increase that has to occur in pO₂ to raise O₂ saturation

What is 2,3-bis-phosphoglycerate?

Where is it made? released?

Where does it bind?

In which Hb type(s) will you not find 2,3-BPG?

• 2,3-BPG is a negative effector of O₂ binding to Hb

• It is made in all cells but only released by RBCs

• It binds to the histidyl groups of both β-globin chains

• You will not find it in HbF or HbA₂, accounts for ↑ in O₂ affinity

Why does 2, 3-BPG have no effect on HbF?

When does it rise?

What is the effect on the lungs? tissues?

Does increased presence of 2,3-BPG shift curve to left or right?

• HbF has no β-globin chains, Ser replaces His 21

• It rises in hypoxia, lung disease, anemia & high altitude acclimation

• Neglibible, because O₂ is never limiting
• Promotes unloading in tissues where pO₂ is steep part of binding curve

• Shifts curve to the right

What is the succession of globins in human development?

• ζ & ε are made in the yolk sac
• liver become the major source of α chains
• γ chain is the precursor of the β chain, just before partuition the γ chain begins to degrade & is replaced w/ the β chain

What is bilirubin and how does it accumulate?

HbF is 2X the mother's HbA, excess Hb → biliverdin → bilirubin. This is a major cause of neonatal jaundice

What is the Bohr effect?

How does it affect the Hb sat'n curve?

Clinical significance?

• The rise in H⁺ levels from the production of CO₂ and/or from the production of lactate from exercising mm. is a negative effector of O₂ affinity
• CO₂ + H₂O ↔ H₂CO₃ ↔ HCO₃+ H⁺

• This causes a shift to the right

• See in DM, pt freq deeply keto-acidic

What effect does CO₂ have on Hb's affinity for O₂?

Where does CO₂ bind?

What forms?

What's different in glycosylated Hb?

• It decreases affinity (shifts curve to right)

• it binds to terminal -NH₂ groups of both α & β chains (non-enzymatic covalent binding)

• spontaneous formation of carbamino Hb (releases more H⁺) more release of O₂ from HbO₂

• it has a reduced ability to pick up CO₂ and expel it to the lungs

What is the Henderson-Hasselbalch equation?

What happens when pH = pK_a?

What is this called?

• It represents the dissociation of acids
• pH = pK_a + log₁₀ [A^-]/[HA]

• the acid is 50% protonated & 50% unprotonated, buffering power of weak acid is at its strongest

• isoelectric point

What is a zwitterion?

How do you calculate pI?

Why do HbA, HbS & HbC all travel different lengths on an electophoresis gel?

• Either the amino or carboxyl group can be ionized

• take the average of the two pK_a values (net charge is zero)

• they all have a dif aa at one position

Which aa acts as a buffer within the normal pH range?

When would an aa's two ionizable groups (amino & carboxyl) be most likely to be ionized?

What's the significance of this?

• Histidine (imidazole group)

• when pH is most different from the pK  value of each group (this is around pH 6-7, close to physiological pH. 

• at physio pH almost all aas are ionized = impermeable to plasma membrane. Need passive/active transport of ions

What are our 3 major buffers?

What is the physio ratio of HCO₃:CO₂?

Does an acidic pH shift curve to left or right? basic?

• HCO₃^-/CO₂ (note: the ratio is maintained because the buffer is an open system)
• HPO4^_--/HPO₄^-
• Protein/H-protein

• 20:1

• acidic shifts curve to the right, basic to the left

Using the formula

pH = pK + log₁₀ [A^-]/[HA]

if pt. is experiencing acidosis pH = 6.4 what is the ratio of [A^-]/[HA]?

pH = 7.4

pK = 6.4

ratio of [A^-]/[HA] is 10:1 (antilog of 1 = 10)

• Tissues - CO₂ enters RBC, hydrated to form HCO₃^-, which then exits in exchange for Cl^-
• Lungs - HCO₃^- reenters RBC in exchange for Cl^-, dehydrated to form H₂O & CO₂ (exhaled)

What is the purpose of the Cl^-/HCO₃^- exchanger?

• aka the anion exchange protein, it ↑ the membrane permeability by more than 10⁶
• it's an obligatory exchange (without one anion, the other stops)
• Does not consume energy as ATP

What is the chief distinguishing characteristic of an enzyme?

An inorganic associate to an enzyme is ____?

An organic associate to an enzyme is _____?

• Substrate specificity

• cofactor

• coenzyme

What are the 6 critical conditions in which enzymes act within?

1. low substrate concentrations
2. avoidance of stable intermediates
3. low temperatures (compared to industrial norms)
4. near-neutral acid-base conditions
5. low pressure
6. low concentrations of the enzyme itself

What are standard conditions (basis for ΔG°¹?

• 1M initial [all reactants]
• neutral pH at 25°C
• 1 atm pressure (760 mmHg)

Identify the enzyme class:

• Catalyze oxidation-reduction reactions
• Catalyze transfer of C-, N-, or P- containing groups
• Catalyze cleavage of bonds by addition of water
• Catalyze cleavage of C-C, C-S & certain C-N bonds
• Catalyze racemization of optical or geometric isomers
• Catalyze formation of bonds btw carbon & O, S, N coupled to hydrolysis of high-energy phosphates

• Oxidoreductases (reducing agents NADH, GSH, etc.)
• Transferases
• Hydrolases (H₂O)
• Lysases (No H₂O)
• Isomerases (Δes configuration w/o Δing the structure or energetic value
• Ligases (bond formation)

What does it mean if ΔG is negative? positive? value near 0?

• exergonic - energy is released
• endergonic - energy must be introduced into the sytem
• equilibrium - therefore reversible

The following is an example of what type of reaction?

ATP + glutamate + NH₃ →ADP + Pi + glutamine

Coupling an exergonic reaction (ATP → ADP + Pi; -8kcal/mol) to an endergonic reaction (Glutamate + NH₃ →glutamine; +4kcal/mol). Net ΔG = -4 kcal/mol

eg. of glutamine synthetase

What happens to the catalyst in a reaction?

What does a catalyst due to the activation energy?

How would you characterize the transition state?

Where does the substrate bind?

• No change
• lowers it
• unstabele
• enzyme's active site

Name 4 types of models that describe the binding between enzyme & substrate.

1. Lock & key
2. Induced fit (most currently accepted)
3. Complementarity of S & E
4. Loose fit - permits strain on substrate

In the induced fit model, structural adapting occurs btw protein & ___?

A conformational Δ in the protein that makes the binding site more ____ to the ligand permitting ____?

• ligand

• complementary; tighter binding

Hexokinase is an example of an enzyme that changes ___ when substrate is bound?

Shape

A nucleophilic (- charge) or electrophilic (+ charge) group in the active site attacks an oppositely charged group of the substrate, name this process and explain what forms.

What else can exhibit the same process? Give an eg?

• Covalent catalysis; which results in covalent binding btw E & S, forming an unstable intermediate in the rxn sequence

• An enzyme bound coenzyme (= holoenzyme, can also form covalent bonds w/ S); eg. pyridoxal phosphate as a prosthetic group for transaminases

From where do most of our coenzymes stem?

Give egs. of coenzymes that we use frequently

• Water-soluble (B & C) vitamins (B-complex)

• NAD⁺/NADH, NADP⁺/NADPH, ADP/ATP, FAD/FADH₂, Coenzyme A (CoASH)

• Note: present at very low concentrations & very reactive compounds; lipid soluble - A, D, E & K)

What metal cofactor is bivalent & usually associated w/ His?

Metals are required in about __ of all enzymes?

Lewis Acids promote what type of catalysis?

• Zinc

• 2/3

• electrophilic

T or F, enzymes are able to work optimally in a broad range of pH? If false, make the statement true.

False - enzymes work in a narrow pH optima at which activity is maximal (activity falls sharply in other ranges)

What happens to catalytic rates when T is below our normal range and increases?

What about when T is just above our T_n?

• Catalytic rates increase

• loss of catalytic activity ensues: 1st disruption of bonds (3° strucure), eventually denaturation (2° structure)

What is the temperature coefficient?

How does it work?

• Q₁₀

• For every 10°C ↑ there is an ↑ in rxn rate; the converse is true for a drop in temp

Q₁₀ = (v₁₊₁₀) / v₁

v₁ = velocity @ original temp

3 key assumptions of Michaelis-Menten Kinetics include:

1. The ES complex is always working - ie. steady state
2. When S is plentiful, all of E is in the ES complex
3. ?

• Since all of E is is in ES complex, the rate of product formation is V_max.

V_max = k₂[ES]

What happens when v = 50% of v_max according to the equation

v = (v_max * [S]) / (K_m + [S])

• K_m = [S]

In a Lineweaver-Burk plot (inverse of M-M), what intersect the x-axis? y-axis?

What happens when [S] >> K_m?, [S] << K_m?

• x-axis: 1/K_m
• y-axis: 1/v_max

• vel of rxn is zero order (i.e. constant & indep of [S]
• vel of rxn is first order (i.e. prop to [S]

What type of inhibition is it when both the substrate & another mlc go for the same active site?

What happens to K_m? v_max?

• competitive inhibition

• K_m increases; v_max NO change

Note: K_m ↑ b/c more S is needed to achieve 1/2 v_max; v_max can be achieved @ high [S] so no Δ

What is the principle behind drugs that use competitive enzyme inhibition to tx disease?

Give 2 examples.

• Analogues of the normal substrate

• Statins - block rate-limiting enzyme of cholesterol synthesis (hypercholesterolemia / atherosclerosis)

• Allopurinol - blocks uric acid production (gout)

What type of inhibition involves separate sites for the binding of substrate & inhibitor?

What happens when I bind?

What happens to K_m? v_max?

• non-competitive inhibition

• rate of catalysis is diminished

• K_m = constant; v_max = ↓

Note: can't be overcome by ↑ in [S], so v_max ↓; binding of enzyme to substrate is not affected, so K_m no Δ

What type of inhibition involves permanent adduct formation?

Give 2 examples.

• uncompetitive inhibition

• organophosphates (pesticides) bind permanently to AchE
• inhibition of Cyclo-oxygenases (COX 1 & 2) by Aspirin (1-constitutive; 2-inducible i.e. in acute response); Aspirin binds to a serine residue at active site (Ser, Thr, Tyr very impt)

What is a homotropic effector?

What is a heterotropic effector?

• when the substrate itself serves as an effector (eg. Hb & O₂

• effector is different from the substate (eg. PFK-1 inhibited by citrate, which is not S for the E

What happens to Km when you have a positive (activation) of an allosteric enzyme?

K_m ↓ (affinity ↑)

Metabolic pathways can either be under regulatory control or have no allosteric control, are the sequences unidirectional or bi-directional?

• unidirectional

Give an example of an energy (ATP) yielding pathway?

Give an example of an energy (ATP) utilizing pathway?

• Catabolic - glycolysis, TCA, fatty-acid oxidation

• Anabolic - gluconeogenesis, cholesterol & fatty acid synthesis

Regulatory rxns are virtually always endergonic or exergonic? and irreversible or reversible?

Why is negative feedback necessary?

• exergonic, irreversible

• b/c uncontrolled fxn places a large drain on certain types of mlcs (eg ATP) need to conserve

What happens at high energy charge? low energy charge?

• High EC: ↑ anabolic pathways, plenty of ATP (use it to make stuff), catabolic inhibited
• Low EC: ↑ catabolic pathways, inhibit ATP-consuming (anabolic)

Diphtheria toxin covalently modifies what type of residue on Euk translocation factor eEF2?

• critical histidyl

Note: NAD⁺ donates ADP-ribose to specially modified histidyl residue on eEF2 (irreversibly blocks cellular protein synthesis)

In cholera, the mechanism of action locks what into a permanently active state?

What happens as a result?

• Adenylate cyclase

• 2nd messenger [cAMP] is kept high → secretion of much Cl^-, other ions →large H₂O loss ("pumping" of H₂O), diarrhea, dehydration →untx - death

Phosphorylation/dephosphorylation is a type of covalent modification, which aas are usually involved?

• Ser & Thr (sometime Tyr) b/c they have reactive (-OH) groups

Note: addition of phosphate is very exergonic but is offset through a phosphatase rxn that is also exergonic

What are the first 2 muscle proteins & cardiac enzymes to appear in plasma following an MI?

• Troponin- most reliable & accurate & Creatine Kinase - (CK2) specific for cardiac infarct (1st appears 8 hrs later, peak @ 24h)

Note: CK2 is an indicator that cardiac not skeletal m. is injured

What isoenzyme appears in the plasma ˜36-48 hrs after MI?

Lactate dehydrogenase

Note: glutamate-oxaloacetate transaminase (GOT) will also appear in plasma

In Pheynylketonuria you have excess ___ due to deficiency of ___?

This prevents production of ___?

Abnormal metabolites build up from phenylpyruvate excess, name them (2). Clinical significance?

Lack of melanin production leads to ___?

• phenylalanine; phenylalanine hydroxylase
• tyrosine (hence no tyr derivatives - melanin, catecholamines, T3/T4)
• phenylacetate & phenyllactate; CNS (MR) & cardiac defects
• albinism

What is feedforward activation? Give eg in glycolysis.

What is feedback inhibition? Give eg in glycolysis.

• The activation of an enzyme by a precursor of the substrate of that enzyme; eg. F-1,6-BP on PK

• Inhibition of an enzyme that catalyzes the production of a substrate by the substrate itself when it has accumulated to a certain level; ATP inhibits its own formation & can inhibit PFK & PK

• Which organ relies 1° on glucose as its fuel source?

• What are the two major glycolytic tissues?

• What is the liver's role in regards to glucose?

• What can generate ATP in anaerobic state & transition to aerobic metabolism?

• Which structure generates ATP & reducing power?

• the brain

• RBC & CNS

• to buffer wide fluctuations in plasma glucose

• skeletal muscle

• RBCs

There is a low permeability to glucose given this statement how does the cell acquire extracellular glucose?

1. Facilitated transport (uniport); GLUTs - tissue specific
2. Co-transport (symport); SGLT - sodium-glucose linked transporter (2 Na⁺/1 Glu

Name the Glucose Transporter

1. Which GLUT is very insulin dependent?
2. Which GLUT has the lowest k_m, highest affinity; is found in brain & RBCs; & has an ↑ in activity during hypoxic conditions?
3. Which one is insulin independent, depends on [glucose] & is in liver & pancreatic β-cells?
4. Which one is assoc w/ dietary fructose?

1. GLUT-4
2. GLUT-1
3. GLUT-2
4. GLUT-5

1. What is the main adv & disadv of aerobic glycolysis?
2. What is the main adv & disadv of anaerobic glycolysis
3. Why do the intermediates of glycolysis stay inside the cell?

1. ATP output high; dependent on other processes 1 Glu → 30-32 ATPs
2. immediate & local - independent of other processes; ATP output limited 1 Glu → 2 ATPs
3. they are phosphorylated, keeps them w/i the cell @ low concs (1st & last are free to leave cell)

• Glycolysis is an exer/endogonic rxn?

• The 1st 5 steps of glycolysis are known as the ___ phase?

• The last 5 steps are known as the ___ phase?

• overall exergonic

• energy investment phase

• energygeneration phase

• What components make up a nucleoside?

• What components make up a nucleotide?

• base + sugar

• base + sugar + phosphate

Note: ester doesn't qualify as a high energy bond

Glucokinase or Hexokinase

1. Which one has a low K_m & low v_max?
2. Negative feedback by G-6-P?
3. Active in liver/kidney?
4. Consumes 1 ATP?
5. Buffer of blood glucose levels?
6. Active in the RBC/brain/heart?
7. Which curve has allosteric characteristics?
8. Which curve has MMK characteristics?
9. Which one converts Glu → G-6-P?

1. hexokinase
2. hexokinase
3. glucokinase
4. both (PFK-1 also)
5. glucokinase
6. hexokinase
7. glucokinase
8. hexokinase
9. both

Glucokinase

• What's the purpose of a high K_m?

• What's the purpose of a high v_max (& resistance to G-6-P inhibition)?

• What substrate indirectly inhibits GK?

• prevents the enzyme from taking too much glucose out of plasma (protective)

• remain active over wide range of [glucose]

• F-6-P by relocating GK into nucleus & binding w/ GKRP

• What are the three rate-limiting enzymes of glycolysis?

• Which one is the regulatory enzyme, the committed step?

• What are its activators & inhibitors?

• When does this enzyme become the rate-limiting step?

• hexokinase, PFK-1, pyruvate kinase

• PFK-1

• activators: AMP, ADP, F-2,6-BP
• inhibitors: ATP, citrate

• when glycogen is the glucose source (skips 1st step)

• What is the gross profit of glycolysis?
• Which steps make ATP?
• What is the net profit?
• Which steps use ATP?
• What type of phosphorylation takes place
• How many times do you go through the cycle for 1 mlc of glucose?

• 4 ATPs
• 1,3-BPG → 3-PG (x2); PEP → Pyruvate (x2)
• 2 ATPs
• Gluc → G-6-P; F-6-P → F-1,6-BP
• substrate-level
• 2

What are inhibitors of Pyruvate Kinase?

What are activators?

What happens in PK deficiency? inheritance pattern?

What ↑es & what is very low?

What type of inheritance are most RBC enzymopathies? the exception?

Which deficiency presents w/ virtually absent 2,3-BPG & mild hemolysis

• ATP, Acetyl-CoA

• F-1,6-BP

• chronic hemolysis; AR (note: 2nd to G-6-P deh)

• 2,3-BPG; ATP

• AR; phosphoglycerate kinase (ATP maker) x-linked

• BPG mutase

What are the 2 major fates of pyruvate?

1. Anaerobic conversion to lactate
2. Aerobic conversion to CO₂ & H₂O (TCA cycle → oxidative phosphorylation)

The obligate coupling of lactate dehydrogenase to G-3-P deh is to what means?

• NADH produced by G-3-P deh (TDH) must be oxidized (by LDH); LDH recycles the NAD⁺ that TDH needs; linked to cell's HIGH NAD⁺/NADH ratio

Bifunctional Enzyme

Why does protein phosphorylation usually cause enzyme inactivation?

It changes the binding site (conformation change in the enzyme)

What is the Warburg effect?

What happens in the conversion of glucose to G-6-P?

• Glucose uptake & degradation is 10 fold higher than in normal, non-cancerous tissues

• it's converted via a hexokinase variant that is insensitive to G-6-P feedback inhibition

What's the difference in the energy metabolism of the spermatozoon?

What's the advantage?

• It uses fructose instead of glucose

• At allows the skippng of steps in glycolysis, much faster process (GLUT-5)

Hepatic v. Muscle Glycogenoses, describe the differences in regards to lactate.

• H: #1 cause hypoglycemia; accumulation of lactate (lactic acidosis)

• M: Inability to produce lg amnts of lactate → intolerance to vigorous exercise →myoglobinuria

Gluconeogenesis

• Which tissues perform gluconeogenesis?
• What is the deal w/ the kidney?
• Which is the only tissue that has glycerol kinase?
• What are the precursor mlcs?
• Which tissue is most impt in the starving state?

• Liver, Kidney cortex
• the kidney medulla is glycolytic/aerobic
• liver
• pyruvate, lactate, alanine, & glycerol
• liver becasue it the body's sole ketogenic & ureogenic organ

Gluconeogenesis

• What are the 4 enzymes that differ from those used in glycolysis

• Why do we need these enzymes

• Which enzyme is lacking in muscle?

• What is essential for PEP synthesis? what is the aa substrate?

• Which one requires Mg⁺²?

• Which one has the active site in the ER lumen

1. Pyruvate carboxylase (biotin depend (also Vit H, Schiff base), acetyl coA → +)
2. PEP carboxykinase (glucagon → +)
3. F-1,6-BPase (F-2,6-BP (glucagon → -), AMP, ADP → -; ATP → +)
4. G-6-Pase (in liver, kidney cortex & pancreatic β-cell)

• to circumvent the physiologically irreversible rxns of glycolysis

• G-6-Pase

• phosphorylation of pyruvate kinase; serine

• PEPCK

• G-6-Pase

Gluconeogenesis

• What is the energy source for gluconeogenesis?

• Is the overall rxn exer- or endogonic

• How much energy is consumed?

• What is essential for PEP synthesis? what is the aa substrate?

• Is low energy charge unfavorable for gluconeogensis?

• ATP & GTP

• exergonic

• 6 ATP equivalents (1 ATP - PC rxn, 1 GTP - PEPCK, 1 ATP - P-glycerate kinase) x2

• YES, will not occur even at ↓ glucose levels

Gluconeogenesis

• Where do our major precursors enter gluconeogenesis?

• pyruvate (lactate, alanine)
• OAA (Propionyl-CoA)
• G-3-P (glycerol)

Gluconeogenesis

• Deficiencies in liver gluconeogenic enzymes often bring about what condition?
• Alcohol also does this to what effect?

• Alcohol deh  converts EtOH → acetaldehyde
• malate →NADPH (malic enzy)

• What is the circuit run by carbon from muscle glycolysis to liver gluconeogenesis?

• Lactic acidosis
• EtOH consumption → NADH accumulation, lactate ≠ pyruvate → lactic acidosis →fatty liver →hypoglycemia (↑ insulin/no gluconeogenesis) = Taco Bell @ 2am

• flushing, tachycardia, nausea
• ↑NADPH favors lipid production → fatty liver

• the Cori cycle

Gluconeogenesis

• What is an immediate regulation of gluconeogenesis?

• How do liver enzymes undergo lg Δes in activity?

• glucagon produced by α cells of the Islets of the pancrease

• phosphorylation

Gluconeogenesis

• What disease is caused by a deficiency of hepatic G-6-Pase?

• pt presents w/ very deep hypoglycemia, so pt unresponsive to ?

• What builds up w/o conversion of lactate → glucose?

• Fate of G-6-P?

• What are the gross signs of disease?

• von Gierke's disease

• glucagon (note: severe lactic acidosis)

• uric acid (uricemia)

• G-6-P → G-1-P (acquire UMP) → UDP-glucose →glycogen

• ↑ serum lipids, ↑ levels of liver glycogen, xanthomata, abdomen protuberant (note: tx w/ glucose administration)

Glycogen syn

• Is glycogen a solid-state complex or soluble?
• What are the main links? the branch links?
• What tissues store glycogen? purpose?

• solid-state w/ enzymes part of complex
• α-1,4; α-1,6
• liver: ↑ after a meal "buffer"
• muscle: maintain for fight/flight (no role in plasma glucose)

Glycogen syn

• Which intermediate resembles 2,3-BPG but is not in RBCs?

• G-1-6-BP (bound like 2,3-BPG)

Glycogen syn

• Which step is very exergonic & ensures progress to UDP-glucose?

• What is actually  being added to G-1-P

• The hydrolysis of pyrophosphate (note: must use Uracil, no subs)

• UMP

Glycogen syn

• T or F glycogen synthesis can initiate on its own? If F make it true. 

• At what end of the chain are mlcs added?

• If glycogenin is used where is the initial glucose member attached? 

• What enzyme does glycogenin used for addition?

• False, it needs a primer, either a pre-existing chain of glycogen or glycogenin

• non-reducing end

• the -OH group of Tyrosine

• None! it's an autocatalytic, self-glucosylating protein

Glycogen syn

• What phosphorylation state activates glycogen synthase?

• Which is the only shared enzyme between glycogenesis & glycogenolysis

• active in non-phosphorylated (a or D) state

• phosphoglucomutase

Glycogen deg

• Name the 4 things that can happen to G-6-P in the liver.

1. re-conversion back to G-1-P
2. glycolysis & supply of pyruvate for TCA
3. formation of free glucose for release into plasma
4. hexose-mono-phosphate pathway-HMP (pentose shunt)

Name the glycogen storage disease.

1. Type V - def. in glycogen phosphorylase; myoglobinuria, exercise intolerance, appears later in life; good prognosis but renal failure can occur
2. Type II - aka "floppy infant syndrome"; excessive [glycogen] abnormal vacuoles in lysosome; acidic-α-glucosidase def.; any tissue that synthesizes glycogen
3. Type VII - def. in muscle PFK-1 & RBC; avoid strenuous exercise
4. Type I - G-6-Pase def.; fasting hypoglycemia, hyperuricemia, fatty liver; prevents release of free glucose from hepatocyte, except single monomer @ branch point
5. Type IV - branching enzyme def. (no α-1,6-linkages) only straight chain; 1st year of life, dead by age 3; hepatosplenomegaly; glycogen levels normal; cirrhosis
6. Type III - debranching enyzme def.(short outer chains); avoid fasting, good outcome
7. Type 0 - glycogen synthase def. in liver only; fasing hypoglycemia & fasting ketosis, unresponsive to glucagon

1. McArdle Syndrome (skel m.)
2. Pompe Disease
3. Tarui's Disease (muscle)
4. von Gierke Disease (liver)
5. Anderson's Disease
6. Cori's Disease
7. Hepatic disorder

Allosteric ctrl of phosphorylase

• free glucose Δes the active shape of phosphorylase to expose ____-phosphate groups which can then be attacked by phosphorylase a phosphate modifying the enzyme to become the b form.

• seryl

Glycogen

• How many Ca⁺² binding sites does calmodulin have?

• Which aa is esp impt to the binding site?

• What substance can directly actiave phosphorylase b w/o prior activation of a form? (note:this activates glycogen phosphorylase, after parturition or strenuous exercise)

• Four

• His

• 5'AMP

• What are the roles of glucagon (liver) & epinephrine (liver & muscle) in regards to glycogen?

• What's the process?

• What enzyme opposes PKA? where does it bind?

• Directly involved in the activation/inhibition of glycogen synthase or phosphorylase

• G protein activated → adenylate cyclase converts ATP to cAMP → PKA activated → phospho kinase activated → phosphorylase b → a (Pi attaches to seryl) → Active glycogen phosphorylase

• specific phosophoprotein phosphatases; recognize serine phosphate groupings

• Does glycogen exhibit positive or negative feedback?

• Negative feedback

• What are the three 1° sources of pyruvate?

• What are the 1° destinations?

• Glycolysis, aas (Alanine) & oxidation of lactate (via LDH)

1. Oxidative decarboxylation to form acetyl-CoA; forms thioester bond, TCA (aerobic via PDH)
2. Reduction to form Lactate (anaerobic)
3. Carboxylation to OAA, Gluconeogenesis (6 ATP equivs)
4. Transamination to Ala

• What disease has a clinical presentation very similar to FAS - severe motor impairment, long philtrum, etc.?

• Inheritance pattern?

• What's the main problem?

• What is the active state of PDH?

• PDH deficiency

• X-linked (dominant)

• No aerobic pathway for pyruvate, lactate builds up

• unphosphorylated

Note: PDH creates reducing power for ATP production in ETC

Name the 5 steps of PDH forming Acetyl CoA

1. Decarboxylation of pyruvate (c-stripped)
2. C- released as CO₂ → Acetate
3. Activated w/TPP
4. Acetate fitted w/CoA-SH
5. Oxidation → products = Acetyl CoA + NADH

Name the 5 coenzymes of the PDH

Note: 1st 4 are from our water-soluble vitamins

Bonus: What diseases is attributed to a missing thiamin in diet?

1. TPP - Vit B1 - Thiamin
2. FAD - Vit B2
3. NAD - niacin
4. CoA-SH - uses pantothenic acid, Vit B5
5. Lipoic acid (susceptible to arsenic poisoning) *only coenzyme synthesized w/i the body, non-essential

• beriberi

Regulation of PDH

• What are the activators?

• What are the inhibitors

• low EC (ADP), high NAD⁺, Ca⁺², pyruvate

• NADH, Acetyl-CoA, high EC (ATP)

T or F, there is an aerobic route for lactate? explain.

• True, lactate → pyruvate →Acetyl CoA → TCA cycle → CO₂ + H₂O
• bypasses the Cori cycle route (via the liver) when a person is continuously jogging

TCA

• Where does it take place?
• What is the 1° substrate?
• Is it catabolic or anabolic?
• Regulatory enzymes?
• What's the gatekeeper?

• mitochondrial matrix
• Acetyl CoA f/PDH (TPP)\
• BOTH = amphibolic

1. Citrate synthase (prok only)
2. Isocitrate deh (major regulator)
3. α-ketoglutarate deh (NO phosphorylation)

• PDH

TCA

• Aconitase can be inhibited by what metabolic poisons?
• What would prevent IDH binding of isocitrate
• which enzyme is similar to PDH
• which rxn breaks a high-energy thioester bond?
• which is the only enzyme that is membrane bound?

• Zn⁺², fluoro-acetate
• phosphorylation @ critical serine
• α-ketoglutarate deh (same 5 coenzyme req'd) (inhib by arsenic)
• cleavage of succinyl-CoA (GDP → GTP)
• succinate deh (FAD → FADH2 (inhibit by arsenic)

TCA

• How many carbons in each structure?

• (2) - Acetyl CoA
• (4) - OAA, Succinyl-CoA, Succinate, Fumarate, Malate
• (5) - α-KG
• (6) - Citrate, cis-Aconitase, Isocitrate

TCA

• Energy potential from 1 turn?

• 3 CO₂ (1 from PDH)
• 4 NADH (1 from PDH) ˜ 3ATP
• 1 FADH₂ ˜ 2ATPs
• 1 GTP

• 15 ATP (3 from PDH)

TCA

• Activators

• Inhibitors

• High pyruvate, low EC (AMP), High NAD⁺, dephosphorylation state

• High ATP, NADH, Acetyl-CoA, phosphorylated state

• Which system is the most energetically favorable (most exergonic)?
• A. Gluconeogenesis
• B. Glycolysis
• C. TCA cycle

• A. -47.6 kJ/mol
• B. -96.2 kJ/mol
• C. -57.3 kJ/mol

Malate-Aspartate Shuttle

• Cytosolic OAA reduced to Malate (NADH → NAD)
• Malate into mt & oxidized to OAA (NAD → NADH + H⁺)
• OAA transaminated w/Glu to α-KG & Aspartate → out to cytosol → reversal back to OAA & Glu → ready for more shuttling

• Result: e^-,s shuttled to make NADH + H⁺

Glycerophosphate (G3P) shuttle

• G3P shuttled into mt → oxidized to DHAP (FADH₂) → DAP out to cytosol → reduced back to G3P or enters glycolysis 

• Result: e^-,s shuttled to make FADH₂