Biochemistry Exam 3

stages of the citric acid cycle

1. Oxidation of Two-Carbon Sugars

2. Regeneration of Oxaloacetate and Harvesting Energy-Rich Electrons

Oxidation of Two-Carbon Sugars

1. 2 carbons atoms (acetyl CoA) are introduced into the cycle by coupling it to a four-carbon molecule (oxaloacetate) to produce a six-carbon molecule (citrate)

2. Subsequently, citrate is oxidized and decarboxylated in a series of reactions to produce a four-carbon molecule (succinyl CoA) 2 CO2 and 2 NADH

Regeneration of Oxaloacetate and Harvesting Energy-Rich Electrons

1. The four-carbon molecule (succinyl CoA) undergoes a series of reactions to regenerate oxaloacetate (so cycle can continue)

2. During this process, GTP, FADH2, and NADH is produced

Forming of Citrate (C6)

• Enzyme: Citrate Synthase

• Substrates/ Co-factors/ Co-enzymes

  • Oxaloacetate (C4)

  • Acetyal CoA (C2)

  • H2O

• Intermediate: Citryl CoA (C6)

• Products

  • Citrate (C6)

  • CoA

Conversion of Citrate (C6) to Isocitrate (C6)

• Enzyme: Aconitase

• Substrates/ Co-factors/ Co-enzymes

  • Citrate (C6)

  • H2O

• Intermediate: cis-Aconitase (C6)

• Product: Isocitrate (C6)

prochiral molecule

• A tetrahedral carbon bonded to two identical groups and two different groups

• Can become chiral when considering the 3-D arrangements (spatial order of the groups around the chiral carbon)

  • Ex. Citrate

pro-R arm vs. pro-S arm in citrate (CAC)

Aconitase dehydrates the pro-R-arm

• S is counter-clockwise (contains the acetyl group from acetyl CoA)

• R is clockwise

Conversion of Isocitrate (C6) to alpha-ketoglutarate (C5)

• Enzyme: Isocitrate Dehydrogenase

• Substrates/ Co-factors/ Co-enzymes

  • Isocitrate (C6)

  • NAD+

• Intermediate: oxalosuccinate (C6)

• Product:

  • alpha-ketoglutarate (C5)

  • NADH

  • CO2

Conversion of alpha-ketoglutarate (C5) to Succinyl CoA (C4)

Enzyme: alpha-ketoglutarate Dehydrogenase Complex

Substrates/ Co-factors/ Co-enzymes:

• alpha-ketoglutarate (C5)

• NAD+

• CoA

• TPP

• Lipoamide

Products:

• Succinyl CoA (C4)

• NADH

• CO2

Conversion of Succinyl CoA (C4) to Succinate (C4) general

• Enzyme: Succinyl CoA Synthetase

• Substrates/ Co-factors/ Co-enzymes

  • Succinyl CoA (C4)

  • Pi

  • GDP

• Products:

  • Succinate (C4)

  • GTP

  • CoA

Conversion of Succinyl CoA (C4) to Succinate (C4) mechanism

1. Convert Succinyl CoA to Succinyl Phosphate via removal of CoA and attachment of Pi

2. Convert Succinyl CoA to Succinate via removal of phosphate (forming a phosphohistidine on the enzyme)

3. GDP enters the active site

4. Substrate-level phosphorylation of GDP via phosphohistidine to form GTP

Conversion of Succinate (C4) to Fumarate (C4)

• Enzyme: Succinate Dehydrogenase

• Substrates/ Co-factors/ Co-enzymes:

  • Succinate (C4) (symmetrical)

  • FAD

• Product:

  • Fumarate (C4)

  • FADH2

Conversion of Fumarate (C4) to Malate (C4)

• Enzyme: Fumarase

• Substrates/ Co-factors/ Co-enzymes

• Product: Malate (C4)

Conversion of Malate (C4) to Oxaloacetate (C4)

• Enzyme: Malate Dehydrogenase

• Substrates/ Co-factors/ Co-enzymes

  • Malate (C4)

  • NAD+

• Products:

  • Oxaloacetate (C4)

  • NADH

Full Steps of the Citric Acid Cycle

1. Formation of Citrate (C6) by citrate synthase

2. Conversion of Citrate (C6) to Isocitrate (C6) by aconitase

3. Conversion of Isocitrate (C6) to alpha-ketoglutarate (C5) by isocitrate dehydrogenase

4. Conversion of alpha-ketoglutarate (C5) to Succinyl CoA (C4) by the alpha-ketoglutarate dehydrogenase complex

5. Conversion of Succinyl CoA (C4) to Succinate (C4) by succinyl CoA synthase

6. Conversion of Succinate (C4) to Fumarate (C4) by succinate dehydrogenase

7. Conversion of Fumarate (C4) to Malate (C4) by fumarase

8. Conversion of Malate (C4) to Oxaloacetate (C4) by malate dehydrogenase

for each acetyl-CoA entering the citric acid cycle, you produce…?

• 3 NADH + H+

• 2 CO2

• 1 FADH2

• 1 GTP (ATP equivalent)

net production for glycolysis, PDC, and CAC for one molecule of glucose

• 4 ATP (net)

• 10 NADH + H+

• 2 FADH2

• 6 CO2

NAD = ?

Nicotinamide Adenine Dinucleotide

FAD= ?

Flavin Adenine Dinucleotide

Shuttles

how NADH + H+ produced in the cytosol via glycolysis enters the mitochondrial matrix for use in the electron transport chain

Glycerol-3-Phosphate Shuttle general

• Prominent in skeletal muscle

• Enzymes:

  • Cytoplasmic Glycerol-3-phosphate dehydrogenase

    • Mitochondrial Glycerol-3-phosphate dehydrogenase

Glycerol-3-Phosphate Shuttle Mechanism

1. NADH+ H+ is oxidized to NAD+ while DHAP is reduced to Glycerol-3-phosphate by Cytoplasmic Glycerol-3-phosphate dehydrogenase

2. Glycerol-3-phosphate oxidized to DHAP, while FAD is reduced to FADH2 by Mitochondrial Glycerol-3-phosphate dehydrogenase

3. FADH2 transfers its electrons to Coenzyme Q reducing it to QH2 which can enter the electron transport chain

Malate-Aspartate Shuttle general

• Prominent in the heart and liver

• Enzymes:

  • Cytoplasmic Malate Dehydrogenase

  • Mitochondrial Malate Dehydrogenase

  • Cytoplasmic Aspartate Aminotransferase

  • Mitochondrial Aspartate Aminotransferase

  • Malate-alpha-ketoglutarate Antiporter (membrane bound)

  • Glutamate-Aspartate Antiporter (membrane bound)

Malate-Aspartate Shuttle mechanism

1. NADH + H+ is oxidized to NAD+ while oxaloacetate is reduced to malate by Cytoplasmic Malate Dehydrogenase

2. Malate can transverse the inner mitochondrial membrane via Malate-alpha-ketoglutarate Antiporter: Malate goes in, alpha-ketoglutarate goes out

3. Malate (now in the mitochondria matrix) is oxidized back to oxaloacetate as NAD+ is reduced to NADH + H+ by Mitochondrial Malate Dehydrogenase

in order for the Malate-Aspartate Shuttle to keep working…?

alpha-ketoglutarate must be present in the matrix and oxaloacetate must be present in the cytoplasm; done by:

• Mitochondrial Aspartate Aminotransferase

• Glutamate-Aspartate Antiporter

• Cytoplasmic Aspartate Aminotransferase

Mitochondrial Aspartate Aminotransferase

transfers an amino group from glutamate (C5) to oxaloacetate (C4) to produce aspartate (C4) and alpha-ketoglutarate (C5)

how is aspartate shutted out to the cytoplasm…?

via the Glutamate-Aspartate Antiporter

• Aspartate goes out, Glutamate goes in

Cytoplasmic Aspartate Aminotransferase

transfers an amine group from aspartate (C4) and alpha-ketoglutarate (C5) to produce glutamate (C5) to oxaloacetate (C4)

oxidative phosphorylation

electrons from NADH and FADH2 are used to reduce molecular oxygen to water

• accomplished through a number of electron-transfer reactions which take place in a set of membrane proteins known as the electron transport chain , ultimately resulting in the reduction of oxygen

negative reduction potential

the oxidized form of a substance has a lower affinity for electrons than H2

• ex. NADH (strong reducing agent) is more likely to donate electrons and has a negative reduction potential

positive reduction potential

the oxidized form of a substance has a higher affinity for electrons than H2

• ex. O2 is more likely to accept electrons and has a positive reduction potential

what is the driving force of oxidative phosphorylation?

the electron-transfer potential of NADH or FADH2

• the electrons always flow to components with more positive reduction potential

electron transport chain types of reactions and carriers

electron flow from NADH (or FADH2) to O2 is accomplished by a series of sequential oxidation-reduction reactions carried out by numerous electron carriers

• FMN

• Iron-sulfur proteins

• Heme-containing proteins

• Cu+ center containing proteins

• Coenzyme Q

Coenzyme Q

• Ubiquinone (Q)- fully oxidized form

• Semiquinone, Radical- the addition of one electron

• Semiquinone (QH)- the addition of one electron and proton

• Ubiquinol (QH2)- fully reduced form

Electron Transport Chain structure & flow

electron flow from NADH to O2 occurs through a chain of large, transmembrane complexes, three of which are coupled to transporting protons (from the matrix to the inner mitochondrial space)

• the electrons always flow to components with more positive reduction potentials (a higher electron affinity)

Complex I- NADH-Q Oxidoreductase (NADH dehydrogenase) general

• Size: 46 polypeptide chains

• Prosthetic groups

  • FMN

  • iron-sulfur clusters

• flow of 2 electrons from NADH to QH2 though this complex pumps out 4 protons

Complex I- NADH-Q Oxidoreductase (NADH dehydrogenase) mechanism

1. NADH is oxidized to NAD+ as FMN is reduced to FMNH2

2. FMNH2 donates electrons (one at a time) to iron-sulfur cluster proteins

3. Iron-sulfur proteins donate electrons (one at a time) to Q to produce QH2

4. QH2 leaves the complex (travels to Complex III)

Complex II - Succinate-Q reductase (Succinate dehydrogenase) general

• Size: 4 polypeptide chains

• Prosthetic groups

  • FAD

  • iron-sulfur clusters

  • Cytochrome b560 (for anchoring)

• The flow of 2 electrons from FADH2 to QH2 through this complex DOES NOT pump out any protons

Complex II - Succinate-Q reductase (Succinate dehydrogenase) mechanism

1. FADH2 donates electrons (one at a time) to iron-sulfur cluster proteins

   1. Succinate reduces FAD to produce FADH2 in the citric acid cycle

2. Iron-sulfur proteins donate electrons (one at a time) to Q to produce QH2

3. QH2 leaves the complex (travels to Complex III)

Complex III- Q-Cytochrome c Oxidoreductase (Cytochrome c reductase) general

• Size: 11 polypeptide chains

• Prosthetic groups

  • Heme (cytochrome) bH (b566) and bL (b562)

• The flow of 2 electrons from “one” QH2 to Cyt c through this complex pumps out 4 protons

Complex III- Q-Cytochrome c Oxidoreductase (Cytochrome c reductase) mechanism

1. When QH2 is oxidized, it donates 2 electrons into the complex

   1. One electron is sent to an iron-sulfur cluster, then to Cyt c1 and finally to Cyt c: 2 protons (from QH2) are transported out

   2. The remaining Q (ubiquinone) becomes part of the “Q pool”

   3. One e is sent to Cyt bH (b566), then to Cyt bL (b562), and finally back to Q (from the “Q pool”) to generate Q-e-, the semi-quinone radical: 2 protons are pumped out

2. A second QH2 enters the complex and is oxidized, delivering 2 electrons to the complex

   1. One electron is sent to an iron-sulfur cluster, then to Cyt c1, and finally to Cyt c : 2 protons are transported out

   2. The remaining Q (ubiquinone) becomes part of the “Q pool”

   3. One electron is sent to Cyt bH (b566) and then to Cyt bL (b562), however the electron sent transported to the Q-e- (from the first half of the Q cycle). The Q-e- is reduced to QH2 by accepting 2 protons from the matrix

overal reactions for complex 3

1. QH2 + 2 Cyt c (Fe3+) + 2H+in → Q + 2 Cyt c (Fe2+) + 4 H+out

2. (2 QH2 + Q + 2 Cyt c (Fe3+) + 2 H+in → 2 Q + QH2 + 2 Cyt c (Fe2+) + 4 H+ out

Complex IV- Cytochrome c Oxidase general

• Size: 13 polypeptide chains

• Prosthetic groups

  • Heme a and a3

  • Cu-containing proteins CuA and CuB

• Flow of 2 electrons from 2 Cyt c to O2 through this complex pumps out 2 protons

Complex IV- Cytochrome c Oxidase mechanism

1. 2 Cyt c donate an electron each to separate Cu-containing peptide units of the CuA dimer (CuA/CuU)

   1. One electron from CuA reduces a Heme a

   2. Heme a reduces Heme a3

   3. Heme a3 reduces CuB

   4. One electron from CuA reduces a Heme a

   5. Heme a reduces Heme a3

   6. However, since CuB can only accept one electron, the 2nd electron stays bound to Heme a3 producing 1 reduced Heme a3 and 1 reduced CuB peptide

2. The reduced Heme a3-CuB peptide can now bind and reduce O2 forming a “peroxide bridge”

3. Two more Cyt c donate an electron (each) to 1 Cu-containing peptide (CuA) all the way to the Heme a3-CuB peroxide bridge; the addition of 2 electrons to the peroxide bridge cleaves the bridge; then, protonation forms Heme a3-(OH) and CuB-(OH)

4. The uptake of 2 protons from the matrix (total of 4 protons) releases the -OH groups as 2 H2O

total production for complex IV

• 4 protons from the matrix are used to produce H2O

• 4 protons from the matrix are pumped out

overall reactions for complex for (actual and per ONE NADH or FADH2)

Actual: 4 Cyt c (Fe2+) + 8H+in + O2 → 4 Cyt c (Fe3+) + 2 H2O + 4 H+out

halved per one NADH or FADH2

the transfer of protons pumped out from the matrix…?

generate a proton-motive gradient

what drives ATP synthesis?

the flow of protons back to the matrix side through ATP synthase

ATP Synthase general structure

• F0 subunit: hydrophobic, in inner mitochondrial membrane

  • c-subunits

  • a-subunits

• F1 subunit: hydrophillic, in mitochondrial matrix

• Two b peptides (b2) connect F0 and F1 together

c-subunits

make of the proton channel component of F0, consisting of a ring of 10-14 c-subunits

• each consists of two alpha-helixes and an aspartic acid residue (Asp)

• connect to the y and e subunits of F1

a-subunit

located just outside of the c-subunit ring of F0

• has two hydrophilic, half channels: one open to cytosol, one open to matrix

F1 subunit

• Size: 85 A in diameter

• Subunits: (alpha3, beta3, upsilon, mu, epsilon)

• alpha3beta3 hexameric ring consisting of alternating alpha and beta subunits with a upsilon-subunit core

• Central stalk consisting of upsilon and epsilon subunits, connects the F1 subunit to the F0 subunit, c-subunit ring

• Each beta-subunit interacts with a different part of the upsilon core

Overall Reaction of ATP Synthase

ADP³- + HPO4²- + H+ → ATP^4- + H2O

Steps of ATP Synthase

1. A single proton from the inner mitochondrial membrane space (cytosol) enters the ATP synthase complex via the F0 alpha-subunit, hydrophilic, cytoplasmic half-channel

2. The proton in the F0, a-subunit, hydrophilic, half-channel protonates Asp 61 on one of the c-subunits of the c-subunit ring

3. With the Asp 61 charge now neutralized, the uncharged Asp 61 can be exposed to the hydrophobic interior of the inner mitochondrial membrane when the c-subunit ring rotates, allowing the next c-subunit to be protonated by a proton entering the F0, a-subunit, hydrophilic, cytoplasmic half-channel

4. Eventually, all of the c-subunits of the c-subunit ring become protonated (uncharged). When these rotate back to the F0 a-subunit, the Asp-61 becomes deprotonated, releasing the proton to the mitochondrial matrix via the other F0, a-subunit, hydrophilic, matrix half-channel

5. The upsilon-subunit core rotates (spins) within the alpha3beta3 hexamer for the F1 subunit

6. Since the c-subunit ring is connected to the upsilon-subunit, as protons enter the c-subunit ring causing it to rotate, the upsilon-subunit core also rotates

7. The upsilon-subunit core has 3 different, interactive surfaces that convert the beta-subunits in the alpha3beta3 hexamer to adopt 1 of 3 different, yet specific confirmations

alpha3beta3 hexamer possible confirmations

1. O-conformation (2nd half): ADP and Pi enter the active site of the beta-subunit

2. L-conformation: ADP and Pi are loosely bound to the beta-subunit in the L conformation

3. T-conformation (1st half): ADP and Pi are tightly bound to the beta-subunit

4. T-conformation (2nd half): ADP and Pi are converted into ATP

5. O-conformation (1st half): the beta-subunit can release ATP

how do we estimate the number of protons needed to make one molecule?

consider the number of c-subunits in the c-ring and divide by 3 ATP

number of ATP =?

number of protons/number of protons/ number of ATP

the beta-subunit can also…?

use ATP to rotate the upsilon-subunit but in the opposite direction of ATP synthesis

most signal transduction pathways (in general) involve the…

1. release of a primary messenger molecule

2. reception of the primary messenger molecule

3. relay of information by a secondary messenger molecule(s)

4. activation of effectors that directly alter the physiological response and

5. termination of the signal(s)

epinephrine

• secreted from the adrenal medulla in response to stress

• stimulates glucose production via gluconeogenesis and glycogenolysis

• binds to the alpha-adrenergic receptor in the liver and the beta-adrenergic receptor in the skeletal muscle

glucagon

• secreted by alpha-cells in the pancreas in response to low glucose levels

• stimulates glucose production via gluconeogensis and glycogenolysis

• binds to the glucagon receptor in liver

insulin

• secreted by beta-cells in the pancreas in response to elevated glucose levels

• binds to the insulin receptor in a variety of tissues to stimulate glucose uptake and glycogenesis

glycolysis

metabolism of glucose

gluconeogenesis

synthesis of glucose

glycogenolysis

metabolism of glucose

glycogenesis

synthesis of glycogen

adrenergic receptor

a seven-transmembrane (7TM) helix receptor

alpha1 receptor

Location: smooth muscle, liver

2nd messenger: Gq

Mechanism of 2nd: IP3 increase, Ca2+ increase

alpha2 receptor

Location: smooth muscle

2nd messenger: Gi

Mechanism of 2nd: decrease cAMP

beta1 receptor

Location: heart, kidney

2nd messenger: Gs

Mechanism of 2nd: increase cAMP

beta2 receptor

Location: smooth muscle, skeletal muscle

2nd messenger: Gs

Mechanism of 2nd: increase cAMP

glucagon receptor

seven-transmembrane (7TM) helix receptor

insulin receptor

a tyrosine kinase receptor

common secondary messenger molecules

• cAMP/cGMP

• Ca2+

• IP3

• Diacylglycerol (DAG)

skeletal muscle

Epinephrine: beta-adrenergic receptors. (cAMP/PKA)

Glucagon: N/A

Insulin: PIP3/Akt (PKB)

liver

Epinephrine: alpha-adrenergic receptors (IP3/Ca2+)

Glucagon: cAMP/PKA, IP3/Ca2+/PKC

Insulin: PIP3/Akt (PKB)

adipose

Epinephrine: alpha-adrenergic receptors (IP3/Ca2+) & beta-adrenergic receptors (cAMP/PKA)

Glucagon: IP3/Ca2+/PKC & cAMP/PKA

Insulin: PIP3/Akt (PKB)

cAMP/Protein Kinase A pathway general and components

• glucagon (liver) and epinephrine (skeletal muscle)

• Components

  • 7TM helix receptor

  • G-protein complex (bind GDP and GTP)

  • Adenylate Cyclase

  • cAMP

  • Protein Kinase A

cAMP/PKA mechanism steps 1-4

• Without a bound ligand, the receptor is bound to a G-protein complex that is “OFF”

• When a ligand binds to the receptor, the Galphas release GDP and binds GTP, turning the GTP-bound alphas-subunit “ON”

• GTP-bound alphas-subunit “ON” stimulates adenylate cyclase

• Adenylate cyclase converts ATP to cAMP

cAMP/PKA pathway mechanism steps 6-11

• Elevated levels of cAMP activate Protein Kinase A (PKA)

• Upon binding, cAMP, the R subunits release the C-subunits

• Phosphodiesterase hydrolyzes cAMP to AMP, which allows the R and C subunits to reassociate, PKA is inactivated

cAMP/PKA pathway mechanism steps 10-12

• The alphas-subunit has GTP-ase activity that will hydrolyze GTP (to produce GDP and Pi)

• The GDP-bound alphas-subunit can now reassociate with the beta/upsilon

• The reformed heterotrimeric G-protein complex can now reassociate with the beta-adrenergic receptor

IP3/Ca2+/Protein Kinase C pathway location and general

Location: glucagon (liver) and epinephrine (liver)

Components:

• 7TM Helix Receptor

• G-protein complex

• Phospholipase C

• diacylglycerol (DAG)

• inosital-1,4,5-triphosphate (IP3)

• Protein Kinase C (PKC)

IP3/Ca2+/Protein Kinase C pathway mechanism

• When a ligand binds to the receptor, the G-protein releases the GDP and binds GTP. The GTP-bound-alpha-subunit dissociates from the receptor and stimulates Phospholipase C

• Phospholipase C hydrolyzes phosphotidylinostiol-4,5-biphosphate (PIP2) to diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3)

• IP3 directly causes a rapid release of Ca2+ by opening IP3-regulated calcium ion channels in the endoplasmic reticulum

• DAG and IP3 activate PKC which will phosphorylate the target proteins (Ser and Thr)

Insulin receptor pathway

Insulin + Insulin receptor.→ activated receptor → Phosphorylated IRS proteins → Localized phosphoinositide 3-kinase → Phosphotidylinositol-3,4,5 -trisphosphate (PIP3) → Activated PIP3-dependent protein kinase → Activated Akt protein kinase → Increased glucose transporter on cell surface

what regulates glycolysis?

glucose transporters

• GLUT1 & GLUT3

• GLUT2

• GLUT4

• GLUT5

GLUT1 & GLUT3

• Found in almost all mammalian tissues

• Used for basal glucose uptake (Km= 1mM)

GLUT2

• found in liver and pancreatic beta-cells

• Km= 15-20 mM

GLUT4

• Found in muscle and fat cells

• Km= 5 mM

• Insulin stimulates the release of vesicles of pre-made GLUT4

• Endurance training increases the amount of GLUT4 present

GLUT5

• found in small intestine

• fructose transporter

regulators of glycolysis in skeletal muscle

• Hexokinase

• Phosphofructokinase (PFK)

• Pyruvate Kinase

hexokinase

high glucose-6-phosphate allosterically inhibits hexokinase

phosphofructokinase (PFK)

• high ATP allosterically inhibits PFK by lowering PFK’s affinity for fructose-6-phosphate; AMP competes with ATP for binding at this allosteric site to relieve ATP’s inhibitory action

• ATP/AMP ratio

a high ATP/AMP ratio…?

PFK is activity is decreased

a low ATP/AMP rato…?

activity is increased

a decrease in pH…?

inhibits PFK

pyruvate kinase

• high ATP allosterically inhibits pyruvate kinase

• fructose-1,6-bisphosphate stimulates pyruvate kinase

• Alanine inhibits pyruvate kinase

glucose-alanine cycle

occurs when muscles are under intense stress, such as fasting (low glucose) or due to prolonged exercise

• since glucose levels are low, alanine inhibits glycolysis

• muscle proteins are converted amino acids, some are used for gluconeogenesis

regulation of glycolysis in the liver

• Glucokinase

• Phosphofructokinase-1

• Pyruvate Kinase

glucokinase

the primary enzyme responsible for phosphorylating glucose in the liver (not hexokinase

• has 50-fold lower affinity for glucose than hexokinase

• Fructose-6-phosphate inhibits glucokinase by lowering the enzyme’s affinity for glucose