Exam 2: Biochemistry Flashcards

Question

eIF2 Regulation

Answer 1

Activated by **guanine nucleotide exchange factor**. Replaces GDP with GTP. Inactivated by a **GTPase**. Under conditions of **stress**, several kinases _phosphorylate eIF2_. **Phosphorylated eIF2** cannot bind gunine nucleotide exchange factor. Low [ternary complex] ⇒ increased activation of amino synthesis genes.

Answer 2

Mechanistic Target of Rapamycin **Serine/threonine kinase.** Major growth factor and nutrient sensor in the cell. Able to modulate translational activity. **Activated via phosphorylation** by **insulin or IGF-1** via **protein kinase B (Akt)** pathway. Also activated by nutrients, especially **leucine**.

Answer 3

* _Resources are plentiful_ * ***mTOR*** _activated_ by ***protein kinase B* (Akt) pathway** * Initiated by **Insulin or IGF-1, leucine** * Activated mTOR _phosphorylates_ **4E-BP1** (eIF4 binding protein) * Phosphorylated 4E-BP1 **releases eIF4E** which can join the eIF4 complex * **Translation proceeds** * _Resources are scarce_ * ↑ [AMP] ⇒ **AMP kinase** activation * AMPK _inhibits_ **mTOR** * **4E-BP1** remains _unphosphorylated_ * **eIF4E** remains _sequestered_ by 4E-BP1 * **Translation is blocked**

Answer 4

Depends on formation of **peptide bond**. Aided by **G-protein elongation factors** (EFs). Requires **2 ATP** (charge tRNA) and **2 GTP** (EFs) per cycle. **_4-step repetitive process:_** 1. **Charged tRNA** brought into **A site** * Aminoacyl-tRNA recruited by **eEF1-GTP** * eEF1-GTP hydrolyzed by **GTPase** to eEF1-GDP and recycled. 2. Amino acid **attached** to nascent polypeptide chain located in the **P site** by ribosomal **peptidyl transferase** * Ribozyme component of large ribosomal subunit 3. Ribosome advances 3 nucleotides towards 3' end of mRNA ⇒ **translocation** * Promoted by GTP-dependent **eEF2** * Target for diphtheria toxin 4. **Empty tRNA** moved to the **E-site**

Answer 5

1. **Release factors (RFs)** recognize the _stop codon_ and bind to the **A site** mimicking a tRNA 2. **Peptidyl transferase** _hydrolyzes_ the completed **peptide chain** from the final tRNA in the **P site** 3. Hydrolysis of **two GTP ⇒ GDP** 4. **RFs** use energy from GTP hydrolysis to induce **conformation change** in ribosome causing **release of nascent polypeptide** through **exit tunnel** 5. mRNA released 6. Large and small ribosomal subunits dissociate

Answer 6

**Co-translational** vs **Post-translational** Can occur in the cytoplasm or other cellular organelles. * **Protein folding** * **Covalent modifications** * Attachment of sugars, phosphate groups, or lipids * Linked by disulfide bridge * **Cleavage** * Multiple subunits **linked** into quaternary structure

Answer 7

Determined mostly by **primary sequence** of the peptide. Primarily involves **non-covalent interactions** between AA side chains: Hydrogen bonding Van der Waals interactions Electrostatic interactions Native-like secondary structures assemble into domains ⇒ **molten globules**. Aided by **chaperones**.

Answer 8

* Binds to **"sticky" hydrophobic patches** on nascent polypeptide chains. * Prevents **non-productive folding** pathways or **aggregation**. * Helps protein to fold by stabilizing intermediate conformations. * Helps with creation of complexes. * Stabilizes proteins as they move through intracellular organelles. * Ex. **Heat shock proteins**

Answer 9

Formed between the **thiol groups** of **cysteine** residues as proteins fold in the **RER**. Catalyzed by ***protein disulfide isomerase***. **Intramolecular disulfide bonds:** * Within a single polypeptide chain * Usually stabilize the tertiary structure **Intermolecular disulfide bonds:** * Between seperate polypeptide chains * Usually stabilize quarternary structure

Answer 10

UPR normally inhibited when chaperone supply adequate. **"Extra" chaperones** bind to **sensing receptors** in **ER lumen**. When **[misfolded proteins] ↑** or too many proteins made, **"extra" chaperones dissociate from sensing receptors** for use in the cell. **Sensing receptors become activated.** Unfolded protein response begins: * **↓ protein synthesis** * **↑ chaperone production** ⇒ ↑ capacity of ER to fold proteins * Continued problem w/ refolding ⇒ **aggregation/accumulation** of abnormal proteins ⇒ **destruction** of unfolded proteins * Failure to resolve high levels of unfolded proteins ⇒ **apoptosis**

Answer 11

Important step in the **maturation processing** of many proteins. **Proteases** cleave by hydrolysis reaction. * **Removal of N-terminus signal sequence** * Cleaved by ***signal peptide peptidase*** * Associated wih the translocon * **Removal of N-terminus initiator methionine** * Allows for addition of acetyl group or fatty acid chains * **Cleavage of precursor proteins** * Activates zymogens

Answer 12

* On **RER**: Insulin synthesized as **preproinsulin** * In **ER**: **met_i and signal sequence** removed converting to **proinsulin** * Proinsulin folds, stabilized by **two interchain disulfide bonds** * In **Golgi**, proinsulin packaged into vesicles * In **vesicles**: **internal sequence** removed (C peptide or connecting sequence) * Regulated secretion of **insulin and C peptide**

Answer 13

* ↑ solubility to prevent agglutination * Protects against proteolysis * Helps folding and oligomerization * Role in cellular sorting * Recognition and antigenicity

Answer 14

Carb attached to **N-atom** in side chain of **asparagine**. Proteins mostly **remain in ER or go to Golgi for expor**t. * **asn-X-thr/ser motif** * **14 sugar** residue transferred from **dolichol phosphate** ⇒ **NH2** on Asn en bloc by ***protein-oligosaccharyl transferase*** * On RER and associated w/ protein translocator * **Sugars added/removed** as protein moves ER ⇒ Golgi * Two categories w/ same 5 sugar core: * **Complex oligosaccharides** * diverse sugars * **High-mannose oligosaccharides** * mostly mannose

Answer 15

Carb attached to O-atom in side chain of serine or threonine. Much less common. * Added in the **Golgi** * **GalNAc** is the first sugar * Added **one at a time** by **glycosyltransferases** * Only a few residues * Length varies

Answer 16

* Proteins made by free ribosomes. * Simpler modifications. * Sugar moieties are not premade. * Transferred as a precursor oligosaccharide.

Answer 17

Reversible post-translational +/- of phosphoryl groups. Kinases ⇒ add Phosphatases ⇒ remove Serine/Threonine Kinases Tyrosine Kinases

Answer 18

* Regulate 1/2 life * Papidly degraded proteins usu. regulatory * Transcription factors, signaling molecules, cytokines * Defective/damaged cells removed * Ubiquitin-proteasome and lysosomal pathways

Answer 19

Cytosolic pathway * Selective * ATP dependent * Quantitatively more significant * Polyubiquitinated protein targeted to proteasome * Ubiquitin ⇒ 76 AA polypeptide

Answer 20

* **E1** hydrolyzes ATP and **adenylates ubiquitin** * Adenylated **ubiquitin transferred to E2** via _thioester_ bond * **E3 (Ubiqitin ligases)** recognizes appropriate proteins and **transfers ubiquitin** * **≥ 4 Ubiquitin** added ⇒ proteasome degradation

Answer 21

"stabilizing" AA on N-terminal like Met ↑ 1/2 life "destabilizing" AA on N-terminal like Arg ↓1/2 life

Answer 22

1+ internal PEST sequences target proteins for rapid degradation

Answer 23

* Lysosomes have **non-specific** proteases called **cathepsins** * Acid hydrolases * Degrades mostly ingested proteins * Role in cellular turnover

Answer 24

* ∆F508 deletion * Misfolding ⇒ ubiquitination ⇒ proteasomal degradation

Answer 25

* Simpler with fewer factors * Ribosomal subunits smaller * **Uses ATP to charge tRNA but not for protein synthesis** * **No mRNA modifications or splicing** * **Transcription & translation coupled** * mRNA **polycistronic** * Uses purine-rich **Shine-Dalgarno sequence** in small ribosomal subunit to position mRNA, AUG, and tRNA_i^Met * N-formylmethionine (fMet) is the first AA * Recognized by body as foreign

Answer 26

Oriented using a **Shine-****Delgarno****sequence** * Positions mRNA on small ribosome * Purine-rich * 6-10 bases upstream from initiating AUG

Answer 27

**High concentrations** * Binds S-12 of bacterial small ribosomal subunit * **Interferes with normal binding of fMet-tRNA_i^Met to P site** * **Inhibits translation initiation** **Low concentrations** * Causes **misreading of mRNA** * Results in the wrong AA being inserted Resistant strains of bacterial have altered ribosome that prevent drug binding.

Answer 28

Binds to the small bacterial ribosomal subunit. **Interefers with binding of incoming aminoacyl-tRNA to the A-site.** **Inhibits translation elongation.**

Answer 29

Interacts with large subunit of baterial ribosome. **Sterically hinders the exit tunnel.** Prevents release of nascent polypeptide. In resistant strains, a single base of rRNA **methylated** and drug cannot bind.

Answer 30

**Inhibits peptidyl transferase activity.** Prevents transfer of growing peptide chain onto next AA residue. Affects both **prokaryotic and mitochondrial** protein synthesis. Reserved for severe cases of infection.

Answer 31

* Toxin binds to plasma membrane and enters the cell. * Catalyzes **ADP ribosylation of EF-2** ⇒ **inhibits translocation** activity * **Inhibits elongation phase** of protein synthesis

Answer 32

All translation of nuclear genes **begins on free ribosomes** in cytosol. Presence of **ER-targeting signal on protein's N-terminus** targets peptide/ribosome pair to ER ⇒ becomes fixed. Location of translation determines where protein will be **ultimately trafficked** and likely **types of protein processing**.

Answer 33

16-30 AA residue at **N-terminus** of protein Has a **hydrophobic core of 6-12 AA** and one or more **positively charged AA** Cleaved upon entry into the ER

Answer 34

Water-filled channel in the ER membrane through which the nascent polypeptide chain passes.

Answer 35

1. Translation **starts on free ribosome** in cytosol 2. **Signal sequence** on N-terminal emerges from ribosome 3. Interacts with **signal recognition particle (SRP)** * Temporarily stops translation ⇒ **elongation arrest** 4. SRP **targets** ribosome-nascent chain complex to a **docking protein** on cytosolic face of RER 5. Upon docking, **GTP ⇒ GDP** promotes **insertion of nascent peptide chain** into **open translocon channel** 6. **SRP released** * Requires GTP hydrolysis 7. Signal sequence removed by ***signal peptidase*** 8. Elongation resumes 9. When translation complete, ribosome released and translocon closes

Answer 36

1. **Resident ER proteins** 2. **Formation of integral/transmembrane proteins** 3. **Default pathway** * No additional ER specific signals * Moved to Golgi for ultimate **constitutive secretion** * Additional tagging can target proteins to lysosomes or regulated secretory pathway

Answer 37

Possess **retrieval signal sequences**. Allows proteins to be retrieved from Golgi and returned to the ER. Most common is **C-terminal KDEL** (Lys-Asp-Glu-Leu) Travel in **COPI coated vesicles.** Ex. Disulfide isomerase

Answer 38

* Assembled at the RER * Majority are targeted and inserted into ER during synthesis * Includes a **stop transfer sequence** * **Transmembrane domain** * hydrophobic core * can be a single domain or cross \>20x * **Translocon** * Can **open the pore** so polypeptide chain translocated into the ER lumen * Can **open laterally** so hydrophobic regions enter the lipid bilayer * Proteins can remain localized in ER membrane or transported in vesicles to other cellular membranes * **Default is the plasma membrane**

Answer 39

* Travel from **ER ⇒ cis face of Golgi** in **COPII coated vesicles** * Move from cis ⇒ trans face to reach **Trans-Golgi Network (TGN)** * Various modifications occur during transit * Proteins **sorted** and placed into different vesicles in TGN * Depends on **tagged signal sequences**

Answer 40

* **Completion of N-linked glycosylation** and **O-linked glycosylation** * By ***glycosyl transferases*** * Glycosaminoglycan chains added to core proteins ⇒ **proteoglycans**

Answer 41

* **Mannose-6-phosphate (M6P)** ⇒ **lysosomes** * In **clathrin coated vesicles** * Signals directing to **secretory vesicles** ⇒ **regulated secretory pathway** * In specialized secretory cells * Secreted and plasma membrane proteins **selectively directed** to apical or basolateral domains * In polarized cells * Requires specific signals

Answer 42

Proteins **segregated and concentrated** into **clathrin-coated** vesicles within the **trans-Golgi network** (TGN). **_Constitutive Pathway_** * **Default pathway** * Delivered from **TGN ⇒ plsama membrane** constitutively unless diverted elsewhere or retained in Golgi * Operates in **all cells** * Many soluble proteins * Supplies plasma membrane with new components **_Regulated Secretion Pathway_** * Proteins diverted to **secretory vesicles** * Concentrated and stored * **Extracellular signal** stimulates secretion * In **specialized secretory cells** only * Small molecules can be **actively transported** from cytosol into preformed secretory vesicles via similar mechanism * Ex. Histamine and neurotransmitters * Often bound to **specific macromolecules** to store at high concentration without extra osmotic pressure * Ex. Histamine ↔︎ Proteoglycans

Answer 43

Membrane-bound compartments inside cell. **Major sorting compartment.** Molecules transported from trans-Golgi membrane ⇒ endosomes in **clathrin coated vesicles**. Classified as **early, sorting, or late** depending on stage post-internalization. Endosomes can continue to develop into lysosomes or recycle back to Golgi. Endocytotic vesicles can enter pathway ⇒ lysosomes or return to plasma membrane.

Answer 44

1. Protein undergo **N-linked glycosylation** in ER lumen 2. Terminal mannose residue phosphorylated by ***phosphotransferase*** in Golgi ⇒ **mannose-6-phosphate** 3. M6P recognized by specific **Golgi receptors** 4. Receptor-protein complex buds off in **clathrin-coated vesicle** 5. Vesicle **fuses with endosomes** 6. **Low pH** ⇒ **dissociation** of glycoprotein from M6P receptor 7. Vesicle w/ fully processed lysosomal enzymes **bud off** from endosomes & **fuse with lysosomes**

Answer 45

AR lysosomal storage disease **Deficient** ***phosphotransferase*** ⇒ no M6P tag Material cannot be broken down ⇒ **inclusions**

Answer 46

Proteins made by cytosolic ribosomes: Retained in the **cytosol** (_default pathway_) Targeted to **nucelus, mitochondria, or peroxisomes** by specific signals

Answer 47

**Short stretch of basic amino acid residues.** Ex. Lysine or Arginine Integral to the protein ⇒ **not cleaved**. Usually sequestered until transport required ⇒ conformational change exposes NLS

Answer 48

1. Protein translated by **free ribosomes** 2. **Nuclear localization signal** bound by **importin** 3. Protein-importin complex **docks and translocated** through **nuclear pore complex** 4. Inside the nucleus, **protein released** facilitated by **Ran-GTP** 5. **Ran-GTP** carries importin **back to cytosol** 6. **Hydrolysis of GTP** releases importin

Answer 49

1. **Nuclear export signals (NES)** binds to **exportin** and **Ran-GTP** 2. Trimeric complex transported through **nuclear pore** 3. **Hydrolysis of GTP** in cytosol dissociates complex

Answer 50

Transport through nuclear pore driven by **concentration gradient of Ran-GDP in cytosol and Ran-GTP in nucleus.**

Answer 51

Due to **mutations in the NLS of SRY protein.** SRY unable to enter nucleus ⇒ no testis-developmental signals XY individuals born as phenotypic females

Answer 52

**Amphiphatic alpha helix** * Basic residues on one side * Hydrophobic residues on the other One or more signal sequences direct protein to the co rrect mitochondrial sub-compartment.

Answer 53

1. Protein translated by **free ribosomes** 2. **Chaperones** bind nascent protein maintaining in **unfolded state** * Requires ATP 3. **Matrix-targeting signal sequence** on N-end recognized by translocase in the outer membrane (**TOM complex)** 4. **N-terminus moved into intermembrane space** 5. **Signal sequence** binds translocase in the inner membrane (**TIM complex**) 6. Protein **moved into the matrix** * Protein spans both matrices for a short time 7. **Signal sequence removed** by ***signal peptidase*** in the matrix 8. **Mature protein forms** within the matrix with help of **chaperones** * Requires ATP

Answer 54

**Internal signals**, rather than N-terminal. Some with **stop-transfer signals.** Can target proteins to outer membrane, inner membrane, and inner membrane space.

Answer 55

Peroxisomal proteins **selectively** imported from the cytosol via **transmembrane tansport**. Most with a **peroxisomal targeting sequence (PTS)** at the **C-terminus**⇒ 3-amino acid (**Ser-Lys-Leu**) Some with target sequence at the N-terminus. Import requires **peroxins** Acts as both chaperones and receptor.

Answer 56

AR disorder **Inability to correctly target proteins to matrix of peroxisomes.** Neurological impairment leads to death at an early age.

Answer 57

1. Pinocytosis 2. Receptor-mediated Endocytosis 3. Phagocytosis

Answer 58

**Cell ingests fluids, molecules, and particles.** Performed in virtually every cell type. **Contitutive and nonspecific.** **Vesicles do not contain a clathrin coat.** Process: 1. Substances to be taken in contacts extracellular surface of plasma membrane. 2. Surface becomes indented. 3. Small pinocytoic vesicles dynamically form in the plasma membrane.

Answer 59

1. Cargo protein binds **specific receptors** forming a **clathrin-coated pit** 2. Pit **buds** from plasma membane forming a **coated vesicle** 3. Vesicles **lose their coat** within the cell and **fuse with early endosomes** 1. Early endosomes are the main sorting station 4. Endosomes mature and **pH ↓** 1. Patches of membrane invaginate into lumen forming **intralumenal vesicles ⇒ multivesciular bodies** 5. Ligands **dissociate** from receptors 6. Most receptors **recycled** to plasma membrane via transport vesicles 7. Ligands digested in lysosomes.

Answer 60

Sometimes receptors/ligands can be **transcytosed** to another part of the plasma membrane. Ex. epithelial cells in lamina propria move Ab from basolateral domain to apical domain.

Answer 61

Ex. of receptor mediated endocytosis * **LDL** binds **LDL receptor** on cell surface * LDL/LDL receptor internalized in clathrin-coated pits ⇒ **clathrin-coated vesicles** * Vesicles lose coat and fuse with early endosomes * Low pH in endosome ⇒ dissociation of LDL from receptor * Continue through endosomal pathway to lysosomes * LDL hydrolyzed to free cholesterol

Answer 62

1. Phagocytic cell engulfs pathogen in **phagosome** 2. Phagosome **fuses** with lysosome ⇒ **phagolysosome** * **TACO** (tryptophanaspartate-containing coat protein) coat must be **removed** before fusion * Internalized membrane components recycled 3. Pathogen **digested** in lysosomes Process called **heterophagy**.

Answer 63

ex. TB bacillus **Avoids digestion by preventing TACO coat from being removed.** Can sometimes be killed via autophagy.

Answer 64

Can escape from phagosomes. Bacteria **secretes a protein that destroys phagosome membrane**.

Answer 65

**Degradation pathway for cellular proteins and organelles.** 3 general types: 1. Macroautophagy 2. Microautophagy 3. Chaperone-mediated autophagy

Answer 66

* **portions of cytoplasm or whole organelles** surrounded by a **vacuole** * forms a _double-membraned sac_ ⇒ **autophagosome** * outer membrane fuses with lysosome ⇒ **autolysosome** * inner membrane and contents digested

Answer 67

**Non-specific** cytoplasmic proteins enter via **invagination** of lysosomal membrane.

Answer 68

* Specific proteins with **targeting signals** directed into lysosomes * Aided by **heat-shock chaperone proteins** * Requires **specific receptors** on lysosomal surface * Significant protein degradation mechanism in **liver and kidney** * Activated during **starvation**

Answer 69

**Dysfunctional metabolism of sphingolipids.** Due to defects in autophagy. Accumulation of large amounts of cholesterol and lipids in lysosomes.

Answer 70

**Disposal of excess proteins stored in secretory vacuoles by fusion with lysosomes.** Rare process occuring in few cell types. Happens when it will likely a long time before products needed again. Ex. mammotrophs of anterior pituitary & prolactin.

Answer 71

**Forms rope-like structure** Characteristic banding pattern High tensile strength **Type I, II, and III collagens**

Answer 72

Fibril forming 90% of body collagen Found in: Dermis Tendons / Ligaments Bone Fascia Organ capsules Cornea

Answer 73

**Forms a 3-D mesh** Ex. **Type IV Collagen** ⇒ _anchoring plaques_ in basal lamina which connects to **Type VII Collagen** ⇒ _anchoring fibrils_ of lamina reticularis

Answer 74

**Flexible collagens with interrupted helices.** Bind to fibrils and connects then to ECM. **Collagen types IX and XII**

Answer 75

Triple helix formed of **2 identical α1 chains** and an **α2 chain**. * Rich in **proline and glycine** * Proline ⇒ kinks peptide chain for helix formation * Glycine ⇒ allows tight turns * Select prolines and lysines **hydroxylated** * Site for stabilizing interchain H-bonds * 3-residue repeating motifs * **Gly-Pro-X** * **Gly-X-Hyp** α chains ⇒ triple helix (procollagen) ⇒ tropocollagen ⇒ fibrils ⇒ fibers ⇒ fascicles ⇒ tendons/ligaments

Answer 76

1. **COL genes** **transcribed** ⇒ α1 and α2 chains * mRNA processed * Moved into cytosol via **gated transport** 2. mRNA **translated** by free ribosomes ⇒ makes **preproprotein** * **Signal sequence ↔︎ signal recognition particle** ⇒⇒⇒ RER * Nascent polypeptide enters **translocon** ⇒⇒⇒ RER lumen * Signal peptide cleaved by ***signal peptidase*** * Converts **preproprotein** ⇒ **proprotein** 3. Select **proline and lysine** residues **hydroxylated** by two ascorbate dependent enzymes * Pro ⇒ hydroxyproline by ***proline hydroxylase*** * Lys ⇒ hydroxylysine by ***lysyl hydroxylase*** 4. Select **hydroxy lysines** receive **O-linked glycosylation** with **galactose or glucose** 5. **Triple helix forms** from _C-terminus to N-terminus_ ⇒ **procollagen** * Stabilized by intra- and interchain **hydrogen and disulfide bonds** * Aided by **chaperones** 6. Procollagen packaged into secretory vessicles * Constitutively secreted via **default pathway** 7. In ECM, **hydrophilic N and C-terminal propeptides cleaved** by ***procollagen peptidases*** ⇒ forms **tropocollagen** 8. Tropocollagen **aggregate into fibrils** d/t hydrophobic effect * Fibrils stabilized by ***lysyl oxidase*** * _Copper-requiring_ enzyme * **Cross-links** the fibrils via staggered covalent bonds 9. Fibrils aggregate to form mature collagen fibers

Answer 77

**Dietary Vit C deficiency** **Impaired function of proline and lysyl hydroxylases** * _Initial symptoms:_ * Fatigue, malaise, gum inflammation * _Progressive symptoms:_ * Depression * Swollen and bleeding gums * Loosening or loss of teeth * Eecchymoses * _Secondary iron deficiency anemia_ * Due to blood loss and ↓ nonheme iron absorption * ↑ risk with malabsorption disorders, cancers, end-stange renal disease

Answer 78

90% of cases due to mutations in **COL1A1 or COL1A2 genes.** Results in abnormal collagen structure. * **_Type I OI_** * Mildest form * Due to mutation resulting in **abnormal protein that does not leave ER or form procollagen** * nonsense or frameshift * **_Types II-IV OI_** * More severe * Caused by **missense mutation replacing glycine residues** * Alpha-chains cannot form tight turns * Steric hinderance and bulge in triple helix during procollagen formation * Protein degraded

Answer 79

Due to mutations in either **collagen genes** or mutations in **proline/lysyl hydroxylases**. * Joint hypermobility * Skin hyperextendibility * Atrophic scar formation * Arterial, intestinal, and/or uterine fragility

Answer 80

**Mutation in gene critical for regulating copper level.** X-linked recessive disorder * **Copper accumulates in kidney and intestines** * **Inadequate Cu in other tissues, esp brain** * Impact function of copper-containing enzymes * **Lysyl oxidase** involved cross-linking of collagen fibrils * Sx. * Sparse, brittle, and twisted hair * Failure to thrive * Lack of muscle tone * Seizures * Progressive brain deterioration * Often do not live past 3 y/o

Answer 81

All the chemical changes that are involved in the occurance and continuance of life. Ability to accomplish these changes at constant body temperature requires enzymatic catalysis & thermodynamic coupling of endergonic and exergonic processes.

Answer 82

The series of steps involved in the breakdown or synthesis of major biological constituents. Cycle = pathway that regenerates the initial substrate Catabolic and anabolic pathways linked via ATP.

Answer 83

Degradative (complex to simpler) Oxidative Exergonic ATP generating Often requires NAD⁺ or FAD

Answer 84

Sum of the pathways that are involved in synthesis and growth. Reductive Energy consuming ATP utilization Often requires NADPH

Answer 85

Expression of enthalpic change. Gives the normalized total heat production per unit time.

Answer 86

Measured in a resting state (awake laying still) Energy requirements for: 1. involuntary muscle work 2. maintenance of osmotic gradients 3. maintenance of body temperature 4. turnover and synthesis of cell constiuents

Answer 87

ΔG = ΔH - TΔS H= enthalpy S= entropy T= temp in kelvins The energy change occuring under conditions of constant pressure and temperature. Additive function and independent of pathway. Total ΔG of a process can be expressed as the sum of ΔG changes of individual steps.

Answer 88

Coupling of an exergonic and endergonic reaction so that net ΔG is negative and reaction can occur. A common intermediate must exist. In an enzyme catalyzed reaction, the common intermediate may not be free and may only exist on the enzyme.

Answer 89

The relative affinity of a molecule, atom, or ion for electrons taken under standard conditions where reactants and products are at unit activity (~1 M) then compared to the proton/hydrogen electrode (H+ and ½ H₂) Often expressed as values corrected to pH 7 (Eº') Interpreted as the relative affinity of the system for electrons as compared to that of a proton: * Negative reduction potential indicates a weaker affinity for electrons than a proton. * Positive reduction potential indicates a stronger affinity.

Answer 90

Dependent on the ratio of oxidant to reductant agent concentration in a cell.

Answer 91

E = Eº' + 2.3 RT/nF log [oxidant] / [reductant] R = gas constant F = Faraday constant Relationship of reduction potential to ratio of oxidant to reductant agents in a cell.

Answer 92

Describes a bond which has a large negative standard Gibb's free energy (ΔGº') of hydrolysis Minimum of -7.0 kcal/mol Indicated with a ~

Answer 93

* Contains two phosphoanhydride bonds which have a ΔGº' of hydrolysis of approximately -7.3 kcal/mol each. * Biological utilization of these high energy bonds require the ATP form. * One or both bonds may be used in a reaction. * Phosphoryl group(s) of ATP can be transferred to acceptor molecules to generate activated intermediates for metabolism. * Can function as coenzyme-cosubstrates.

Answer 94

Use of both phosphoanhydride bonds of ATP is the functional equivalent of using 2 ATP's. Reactions coupled to the hydrolysis of the pyrophosphate (PP_i) product to 2 orthophosphates (P_i) by pyrophosphatase which drives reactions forward.

Answer 95

Catalyzes the reversible reaction: AMP + ATP ⇔ 2 ADP

Answer 96

Total concentration of adenine nucleotide pool essentially constant, however, ratio of adenine nucleotides vary with metabolic state of the cell. Concentrations of ATP, ADP, and AMP in a cell are in rapid equilibrium due to activity of adenylate kinase. The cosubstrate pool communicates between and significantly influences the different pathways of the cell which utilizes those cosubstrates. AMP level most sensitive parameter to change in pool and usually initiates the responses to decreased ATP levels.

Answer 97

Energy charge = ½ • ( [ADP] + 2[ATP] ) / ( [AMP] + [ADP] + [ATP]) A stoichiometric expression of the mold fraction of high energy phosphate bonds present relative to the maximal high energy bonds possible. 0 = nucleotides are totally in the form of AMP 1 = nucleotides are totally in the form of ATP

Answer 98

ΔG_ATP→ADP = ΔGº'_ATP→ADP + RT ln ( [ADP] x [P_i] / [ATP] ) Actual ΔG for ATP hydrolysis in a cell is dependent upon and may be calculated from the concentrations of ATP, ADP, and inorganic phosphate. May be very different from the ΔGº'. In a resting cell may be as high as = 15 kcal/mol which is equivalent to an energy charge of approximately 0.9. "High energy charge", "large negative ΔG of ATP hydrolysis", and "resting cell" all indicate that ATP is plentiful.

Answer 99

* Pathways for synthesis and breakdown of the same constituent are never the same, although individual steps may be reversible and utilized in both pathways. * Opposing pathways typically occur in seperate cellular compartents and/or different tissues or organs. * Regulation of pathway enzyme activity by: * product inhibition * allosteric regulation * covalent regulation i.e. phosphorylation * Changes in the rate of synthesis/degradation of an enzyme or its mRNA.

Answer 100

* Glucose transported across cell membranes by: 1. Facilitated diffusion via GLUT transports down their concentration gradients. 2. In renal and intestinal epithelium, transported against its concentration gradient by Na⁺-glucose co-transporters (SGLTs)

Answer 101

* _Glucose-dependent_ tissues such as RBC's and brain have low K_m insulin-independent GLUT1 or GLUT3 transports respectively. * In peripheral tissues such as muscle which are _glucose-independent_, GLUT4 transports have a low KM but is insulin-dependent ⇒ allows cross regulation. * The liver which _does not rely on glucose_ for energy, uses the GLUT2 transporters have a high K_M for glucose but is insulin-independent. * Limits glucose uptake to conditions when blood glucose levels are high. * Allows the transporter to act as a sensor of high blood glucose levels. \* Normal fasting blood glucose levels are 3.9 - 5.5 mmol/L.

Answer 102

* Located in muscle and adipose tissue. * Relatively low K_m for glucose so *would* transport around normal fasting levels. * _Insulin-dependent_ glucose transporter: * When insulin is absent, the transporters are removed from the plasma membrane and sequestered into vesicles. * Functional but not in the membrane. * Insulin signaling stimulates the movement of the transporter from internal stores to the plasma membrane. * _In skeletal muscle_, exercise stimulates GLUT4 translocation to the plasma membrane through AMP-activated protein kinase (AMPK) via unknown mechanism. * Long-term exercise also increases the amount of GLUT4 in the muscle cell.

Answer 103

1. Binding of insulin to the α-subunit of its receptor activates a **tyrosine kinase** domain resulting in auto-cross-phosphorylation of tyrosine residues in the β-subunits. 2. Negative charge of the phosphates causes IRS (insulin receptor substrate) proteins to bind to the β-subunit. 3. IRS proteins phosphorylated at two Tyr residues by the kinase activity of activated insulin receptor. 4. **Phosphorylated-IRS** dissociate from the receptor then bind to and activate proteins with SH2 domains i.e. PI-3-kinase (Phosphatidylinositol-3-kinase). 5. **PI-3-kinase** phosphorylates PIP2 to **PIP3**. 6. PIP3 activates **PDK-1** (phosphoinositide-dependent kinase). 7. PDK-1 activates downstream effectors **Akt** and **PKB** which results in the movement of GLUT4 to the cell surface in adipose and muscle, increasing glucose uptake. Akt/PKB also: * phosphorylates and inactivates GSK3 (glycocen synthase kinase 3) resulting in increased glycogenesis. * Activate amino acid uptake and protein synthesis * Increase lipid synthesis * Inhibit gluconeogenesis * decrease cAMP levels by activating phosphodiesterase * various gene expression modulations both +/- * Increases protein synthesis by activating a kinase (mTOR) that ultimately results in the activation of eIF4 and EF2 from protein translation.

Answer 104

* Phosphorylation of glucose prevents back diffuse out of the cell via the transporter and commits it for use in that cell. * **Hexokinases** * Found in tissues such as muscle and brain. * Has a low K_M for glucose * Can phosphorylate other monosaccharides but affinity for glucose considerably higher * Show product inhibition by glucose-6-phosphate. * **Glucokinase** * Found in liver and pancreatic β-cells * Has a high K_M for glucose * Shows no direct product inhibition

Answer 105

* Despite being monomeric, glucokinase displays sigmoidal kinetics towards glucose. * The inflection point of the glucokinase enzyme curve is such that small changes in blood glucose levels causes significant changes in enzymatic activity. * When blood glucose levels are high the hepatic glucokinase becomes significantly more active. * Hexokinase, however, is fully saturated a normal concentrations of blood glucose.

Answer 106

glucose + 2 NAD⁺ + 2 ADP + 2 P_i → 2 pyruvate + 2 NADH + 2 ATP + 2 H₂O + 2 H⁺

Answer 107

1. Priming steps * Two ATP-linked phosphorylations * Functions to prevent diffusion of the intermediates of the pathway out of the cell. 2. Oxidation/reduction with the production of NADH * Results in the generation of ATP 3. Re-oxidation of the NADH produced.

Answer 108

The synthesis of ATP involving two coupled reactions linked by a common intermediate containing a high-energy bond.

Answer 109

1. Irreversible phosphorylation of _glucose_ at carbon-6 by **hexokinase** using ATP•Mg²⁺ to produce _glucose-6-phosphate_. * Hepatic isozyme is **glucokinase**. * Traps glucose because cell membrane is impermeable to phosphate esters. * Commits glucose to intracellular metabolism but NOT glycolysis. 2. Freely reversible aldose-ketose isomerization of _glucose-6-phosphate_ to _fructose-6-phosphate_ by **phosphoglucose isomerase**. 3. Irreversible phosphorylation of _fructose-6-phosphate_ to _fructose-1,6-bisphosphate_ using ATP by **phosphofructokinase (PFK1)**. * True commitment step and primary regulatory site for glycolysis. 4. _Fructose-1,6-bisphosphate_ split into two triose phosphates: _dihydroxyacetone phosphate (DHAP)_ and _glyceraldehyde-3-phosphate (GAP)_ by **aldolase**. * DHAP freely interconverted to GAP by **triose phosphate isomerase**. * Only GAP enters next stage of glycolysis. 5. **Glyceraldehyde-3-phosphate dehydrogenase** catalyzes the reversible oxidation of _glyceraldehyde-3-phosphate_ to _1,3-bisphosphoglycerate_. * NAD+ converted to NADH \*\* 6. Phosphate from the carboxyl group from _1,3-bisphosphoglycerate_ is reversibly transferred to ADP to produce ATP and _3-phosphoglycerate_ by **3-phosphoglycerate kinase**. * First substrate-level phosphorylation. * Additional mutase found in erythrocytes which transfers carbonyl phosphate of 1,3-bisphosphoglycerate to carbon-2 to produce 2,3-bisphosphoglycerate (2,3-BPG). 7. _3-phosphoglycerate_ converted to _2-phosphoglycerate_ by **phosphoglycerate mutase**. 8. _2-phosphoglycerate_ converted to _phosphoenolpyruvate (PEP)_ by **enolase** with loss of H₂O. 9. Phosphate of _PEP_ is transferred to ADP by **pyruvate kinase (PK)** to produce _pyruvate_ and ATP. * Irreversible * Has a ΔGº' of -14 kcal/mol 10. NADH produced is recycled back to NAD+ * In aerobic conditions via **malate shuttle**. * In anerobic conditions pyruvate converted to lactate via **lactate dehydrogenase**.

Answer 110

Most important regulatory step in glycolysis because: * It is the commitment step for the glycolytic pathway. * It is allosterically modulated by many metabolic intermediates and products.

Answer 111

Allosteric effectors act by influencing the equilibrium between the active and inhibited forms of the enzyme. Binds preferentially to one form or the other and stabilizes that form. * **Activators ⇒** high energy signals * Citrate * ATP * Fructose-2,6-Bisphosphate\*\*\* * **Inhibitors** ⇒ low energy signals * ADP * AMP

Answer 112

Fructose-2,6-bisphosphate (F-2,6-P₂) is a positive heterotropic effector of PFK1. F-2,6-P₂ is synthesized and degraded by a multifunctional enzyme with both: * kinase activity ⇒ **PFK2** * phosphatase activity ⇒ **F-2,6-Bisphosphatase**

Answer 113

Multifunctional enzyme reponsible for synthesis and degradation of F26BP. **PFK2 & F26BPase** Phosphorylation of either domain inhibits its catalytic activity. **Liver:** PFK2 enzyme a substrate for cAMP-dependent PKA. Phosphorylation site for liver PFK2 isozyme lies within the kinase domain. Glucagon ⇒ phosphorylation ⇒ ↓ kinase activity & ↑ phosphatase activity ⇒ ↓ [F26BP] ⇒ ↓ PFK-1 activity **Skeletal Muscle:** PFK2/F26BPase isozyme has no phosphorylation sites. Is not covalently regulated.

Answer 114

* Hepatic PFK1 is primarily regulated by the [F-2,6-BP] * The primary function of F-2,6-BP in the liver is to make PFK1 sensitive to regulation by glucagon and other hormones. * The liver does not consume glucose as fuel during times of need -- rather it makes glucose for use by other tissues.

Answer 115

**_Covalent Regulation_** **(Liver Only)** * _Fasted state:_ * **Glucagon** → cAMP → PKA → _phosphorylated PK_ ⇒ **PK inhibited** * _High glucose:_ * **Insulin** → ↑ phosphatase activity → _dephosphorylation of PK_ ⇒ **PK activation** **_Allosteric Regulation_** **(All cells)** * _Fasted state:_ * **Acetyl CoA** * **Long chain fatty acid** * **Alanine** * _High energy state:_ * **ATP** * **F-1,6-BP**

Answer 116

Glycolysis occurring under anaerobic conditions.

Answer 117

**Pyruvate + NADH → lactate + NAD⁺** Catalyzed by ***lactate dehydrogenase***. Occurs in mammalian tissues specifically skeletal muscle. Allows glycolysis to continue under anerobic conditions. Lactate enters the blood and is ultimately reconverted to glucose in the liver via gluconeogenesis.

Answer 118

Occurs in microorganisms such as yeast. **1. Pyruvate → CO₂ + acetaldehyde** Catalyzed by pyruvate decarboxylase. **2. Acetaldehyde + NADH → ethanol + NAD**⁺ Catalyzed by alcohol dehydrogenase.

Answer 119

1. Electrons are transferred from cytosolic NADH to oxaloacetate forming malate and NAD⁺. 2. Malate enters the mitochondrial inner membrane via malate/α-ketoglutarate transporter. 3. Inside the matrix, malate is reoxidized by malate dehydrogenase and NAD⁺ to form OAA and NADH. 4. OAA is converted to aspartate via a transamination reaction. 5. Aspartate is transported back to the cytosol via a glutamate/aspartate transporter. 6. In the cytosol the aspartate undergoes transamination to reform OAA. \*Shuttle is readily reversible: important in gluconeogenesis.

Answer 120

Couples the cytosolic oxidation of NADH with the mitochondrial reduction of FAD. 1. Cytoplasmic NADH utilized by glycerol-3-phosphate dehydrogenase to convert dihydroxyacetone phosphate to glycerol-3-phosphate. 2. Glycerol-3-phosphate is then converted back to DHAP by mitochondrial version of the dehydrogenase which resides on the inner mitochondrial membrane. FAD is reduced to FADH₂. 3. Electrons from FADH₂ are trasferred to the electron carrier Q which enters the respiratory chain as QH₂.

Answer 121

* Alternate pathway from **glucose-6-phosphate** * Primary function is **generation of NADPH** * Important in neutralization of ROS * Used to support fatty acid biosynthesis (cytosol) * Serves as a source for **ribose-5-phosphate synthesis** * Used in synthesis of nucleic acids * Used to produce **glyceraldehyde-3-phosphate** * Fed into TCA and ETC cycles * Pathway can be divided into two sections: * Oxidative * Non-oxidative

Answer 122

Rate-limiting and regulated step in PPP Stimulated by G6P and NADP⁺ NADPH is a competitive inhibitor

Answer 123

* Most common of all enzyme deficiency-related diseases * X-linked * Cuts off cell's supply of NADPH * Affects RBC's most who cannot produce new proteins * Oxidative stress leads to hemolysis and anemia * Infection most common * Drugs, chemicals, certain foods * Fava beans * Anti-malarials * Certain antibiotics * Naphthalene * Heterozygotes have some resistance against malaria * Therefore gene prevalent in the Mediterranean and Africa

Answer 124

* Glutathione and the glutathione peroxidase system is the principal antioxidant defense system in mammalian cells * Glutathione is a tripeptide which contains a central Cys residue. * Reduced form (GSH) * Oxidized form (GSSG)

Answer 125

* Glucose preferred but cells will also use other sugars * Fructose and galactoase are significant in the diet * Mannose important components of glycoproteins * Metabolism of other sugars are fed into glycolytic pathways * Sugars must be phosphorylated by the cell before they can be used. * Hexokinase/glucokinase can phosphorylate other monnosaccharides but their K_M are significantly higher than glucose's * Galactokinase present in most cells * Fructokinase in liver only

Answer 126

1. *Fructokinase* (found primarily in the liver) phosphorylates _fructose_ at the 1 position producting _fructose-1-phosphate_ * No mechanism exists to convert F-1-P to G-1-P 2. *Aldolase B* (liver isozyme of aldolase) can split both _F-1-P_ and F-1,6-P₂ to form _dihydroxyacetone phosphate (DHAP)_ and _glyceraldehyde_. 3. _Glyceraldehyde_ formed can be: * Phosphorylated by *triose kinase* to form _glyceraldehyde-3-phosphate_ → enter glycolysis * Reduced to _glycerol_ by *alcohol dehydrogenase* then phosphorylated by *glycerol kinase* to form _glycerol-3-phosphate_ which is then converted to _DHAP_ by *glycerol phosphate dehydrogenase*. * DHAP converted to GAP by *isomerase*.

Answer 127

* Due to deficiency of fructokinase * Relatively benign * Leads to accumulation of fructose in the urine

Answer 128

* Autosomal recessive * Absence of aldolase B (liver isoform) * Results in accumulation of fructose-1-P in the liver * Depletes levels of ATP and P_i * Low P_i levels inhibit glycogenolysis * Low ATP levels inhibit gluconeogenesis * Low P_i activates AMP deaminase in muscle * Results in increased purine catabolism and hyperuricemis → gout * Low P_i prevents phosphorylation of ADP so adenylate kinase will convert 2 ADP → ATP + AMP * AMP degraded to urate * Symptoms include: * Vomiting * Hypoglycemia * Jaundice * Metabolic acidosis * Coma

Answer 129

Most tissues are cabable of metabolizing galactose. 1. *Galactokinase* phosphorylates galactose at the 1 position producing _galactose-1-P_. 2. *Galactose-1-phosphate uridylyltransferase* (GALT) switches galactose for glucose from _UDP-glucose_ producing _UDP-galactose_. 3. UDP-galactose can interconverted to _UDP-glucose_ by *UDP-glucose-4-epimerase*.

Answer 130

* Most common cause of galactosemia * Symptoms include: * Failure to thrive * Liver damage * Bleeding * Sepsis * Cataracts - later on * If galactose restricted diet provided within the first 10 days the most severe complications can be avoided: * Neonatal death * Liver failure * Intellectual disability * Children with galactosemia remain at risk for developmental delays and problems with speech and mother function.

Answer 131

* Rare disorder * Morbidity limited to cataract formation

Answer 132

* Derived from ATP and pantothenic acid (Vit B5) * Contains a reactive thiol group (-SH) that is covalently linked to the acetyl group via a thioester bond * Acetyl~CoA readily donates acetyl groups to other acceptors

Answer 133

* Member of the α-ketoacid dehydrogenase family * Large multienzyme complex which contains 3 enzymes in multiple copies * Very efficient due to close spatial proximity of complexes **Enzyme subunits** * **E1**: *pyruvate dehydrogenase* (aka pyruvate decarboxylase) #30 * **E2**: *dihydrolipoyl transacetylase* #60 * Acts as a swinging arm between E1 & CoA preventing substrate from diffusing away ⇒ _substrate channeling_ * **E3**: *dihydrolipoyl dehydrogenase* #12 * Contains two stoichiometric coenzymes/cosubstrates * NAD * CoA * Contains three catalytic coenzyme prosthetic groups * thiamine pyrophosphate (TPP) * FAD * lipoic acid * Contains two regulatory enzymes associated with but not part of the complex * PDH kinase * PDH phosphatase

Answer 134

1. **Pyruvate** is _decarboxylated_ and the **acetyl group** is attatched to thiamine pyrophosphate **(TPP) coenzyme** of ***pyruvate decarboxylase*** (E1). 2. Acetyl group _transferred_ to the **lipoic acid** covalently bound to ***dihydrolipoyl transacetylase*** (E2), 3. The acetyl group, bound as a **thioester** to the side chain of lipoic acid, is transferred to **free CoA**. 4. The **sulfhydryl form of lipoic acid** is _oxidized_ by **FAD**-dependent ***dihydrolipoyl dehydrogenase*** (E3) to regenerate the **oxidized lipoic acid**. 5. **FADH₂** on *E3* is _reoxidized_ to FAD as **NAD⁺** is **reduced** to NADH₂ + H⁺.

Answer 135

**_Allosteric regulation_** * _Inhibitors_ (feedback inhibition) * Acetyl~CoA * NADH **_Covalent regulation_** (main mechanism) * PDH **inactivated by phosphorylation** by ***PDH kinase*** * PDH **activated by dephosphorylation** by ***PDH phosphatase*** **_PDH kinase and PDH phoshatase are in turn allosterically regulated:_** * *PDH kinase* * _activated_ (high-energy signals) * **ATP** * **acetyl-CoA** * **NADH** * _inhibited_ * **pyruvate** * *PDH phosphatase* * activated * ↑ [Ca²⁺] * important in skeletal muscle

Answer 136

Niacin (B₃) → NAD+/NADH Thiamine (B1) → TPP Lack of either will reduce/block activity of PDH complex. ↓ ATP synthesis CNS dependent on ATP ⇒ reduced CNS function

Answer 137

Arsenic in the form of trivalent arsenite (AsO33-) forms a stable complex with thiol groups of lipoic acid. Lipoic acid unable to serve as coenzyme for PDH.

Answer 138

**Acetyl~CoA + 3 NAD⁺ + FAD → 2 CO₂ + 3 NADH + FADH₂** * 8 steps * 4 oxidations that produce NADH or FADH₂ * one substrate-level phosphorylation producing GTP * Except for succinate dehydrogenase which is embedded in the inner mitochondrial membrane all enzymes are located in the matrix

Answer 139

1. **Formation of citrate.** * Condensation of **acetyl~CoA** and **oxaloacetate (OAA)** catalyzed by ***citrate synthase***. * Reaction made **irreversible** by the hydrolysis of the thioester bond of acetyl~CoA. * Facilitated by an enzyme-bound intermediate, citroyl~CoA * **Citrate synthase inhibited by citrate via competitive inhibition** 2. **Isomerization of citrate to isocitrate.** * **Citrate** isomerized to **isocitrate** by ***aconitase***. * ΔGº' = 6.3 kJ/mol but reaction pushed to the right in vivo because product rapidly consumed in next step. 3. **Oxidation of isocitrate to α-ketoglutarate and CO₂.** * **Irreversible** decarboxylation of **isocitrate to α-ketoglutarate** by ***isocitrate dehydrogenase***. * Inhibited by ATP and NADH. * Stimulated by ADP and Ca²⁺ * Loss of CO₂ make the reaction irreversible * **NADH** produced 4. **Oxidation of α-ketoglutarate to succinyl~CoA and CO₂.** * Catalyzed by ***α-ketoglutarate dehydrogenase** complex*. * Same family as PDH dehydrogenase. * Cosubstrates (NAD and CoA) * Coenzymes (TPP, FAD, and lipoic acid) * **Inhibited by succinyl~CoA and NADH** * **Activated by Ca²⁺** * Not regulated by phosphorylation/dephosphorylation. * **NADH** produced * Loss of CO₂makes reaction **irreversible.** 5. **Conversion of succinyl~CoA to succinate.** * *Succinate thiokinase* catalyzes the hydrolysis of the thioester bond in succinyl~CoA paired to the conversion of GDP to GTP. * GTP interconvertible to ATP by *nucleoside diphosphate kinase*. 6. **Oxidation of succinate to fumarate.** * Catalyzed by ***succinate dehydrogenase*** * **FADH₂** made * Enzyme located on inner mitochondrial membrane. * ΔGº' = 0 so reaction can go either way but pushed to the right in vivo because fumarate used in next step. 7. **Regeneration of oxaloacetate.** 1. **Fumarate** converted to **malate** by ***fumarase***. 2. **Malate** converted to **oxaloacetate** by ***malate dehydrogenase*** * NADH made * Rxn has a ΔGº' = 29.7 kJ/mol but pulled to the right because OAA used by citrate synthase in step 1

Answer 140

Regulated almost exclusively at the three irreversible steps. 1. *Citrate synthase* competitively inhibited by citrate. 2. *Isocitrate dehydrogenase* * Inhibited by ATP and NADH * Stimuated by ADP and Ca⁺⁺ 3. *α-ketoglutarate dehydrogenase* 1. Inhibited by succinyl~CoA and NADH 2. Activated by Ca⁺⁺

Answer 141

TCA cycle intermediates can be used for other reactions such as: * amino acid synthesis * fatty acid synthesis * gluconeogenesis

Answer 142

Intermediates of the TCA cycle can be provided for by other metabolic pathways, specifically amino acid metabolism.

Answer 143

Inner mitochondrial membrane bound complex. Consists of four seperate protein complexes. Each complex accepts or donates electrons to/from mobile electron carriers (coenzyme Q and cytochrome C). ETC pumps protons from matrix to intermembrane space to form a proton gradient.

Answer 144

* **Complex 1: NADH dehydrogenase** * Flavin mononucleotide (FMN) coenzyme accepts two electrons from NADH to become FMNH₂. * FMNH₂ transfers electrons to CoQ to form CoQH₂. * Enzyme contains iron-sulfur (Fe-S) centers which acts as intermediate electron carriers. * **Complex 2: Succinate dehydrogenase** * Electrons from succinate of TCA cycle transferred via Fe-S centers to FAD to form FADH₂. * **Complex 3: Cytochrome bc₁ reductase** * Electrons from CoQH₂ (from complex 1) are used to reduce cytochrome c which acts as electron carrier. * Contains Fe-S centers which act as intermediates. * Contains heme group which shifts from Fe³⁺ to Fe²⁺ and back as electrons move through. * **Complex 4: Cytochrome oxidase** * Contains two different heme groups * Heme *a* * Heme *a₃* * Contains two Cu ions which act as intermediates * Electrons from cytochrome c transferred to heme *a* via one of the Cu centers, then from heme *a* to heme *a*₃via other Cu center, and finally to O₂ to form H₂O.

Answer 145

Oxidized form ubiquinone reduced to ubiquinol after accepting electron. Accepts electrons from complex I and II of the ETC.

Answer 146

Contain a heme group in which the iron shifts from Fe³⁺ to Fe²⁺ oxidations states and back as electrons move to and from the heme group. All contain the porphyrin ring with differing side chains.

Answer 147

Measures the ease with which an electron can be added or removed. The more positive the value of E'º, the greater the tendancy of the oxidant in the redox pair to accept electrons. Electrons flow from the redox pair with the more negative E'º to one with a less negative/more positive E'º.

Answer 148

The redox potential (E'º) is related to the free energy change in the reaction by the Farady constant (F). ΔG'º = -*n*FΔEº *n* = number of electrons transferred ΔEº = difference in reduction potentials of the overall reaction

Answer 149

In the cell, the effective reduction potential (E) depends on the concentration of reactants.

Answer 150

Due to the reduction potentials of each complex the electrons always flow downhill.

Answer 151

Used to block electron flow in ETC. Carriers befor the block become reduced and those after remain oxidized.

Answer 152

ATP synthase uses the proton gradient set up by ETC to produce ATP. Dependent on the integrity of the inner mitochondrial membrane. No high-energy intermediate exists (as with substrate-level phosphorylation).

Answer 153

When more electrons enter the ETC than can be immediately passed to O₂ a state of **oxidative stress** exists. Highly reactive superoxide free radicals such as superoxide (•O₂^-) are generated. Mitochondria have systems to eliminate free radicals: 1. Superoxide (•O₂^-) is converted to peroxide (H₂O₂) by *superoxide dismutase.* 2. Peroxide converted to water by *glutathione peroxidase.* NADPH used by *glutathione reductase* to regenerate *glutathione peroxidase.*

Answer 154

The coupling of ATP synthesis and electron transport. Neither process can occur without the other.

Answer 155

The stoichiometry of ATP synthesis relative to the substrate that is oxidized. The amount of ATP formed per ½ O₂ consumed (or per pair of electrons. Approaches 3 for NADH. Closer to 2 for FADH₂ (because succinate oxidation bypasses complex I)

Answer 156

For each pair of electrons that travel through the ETC and transferred to O₂: Complex 1 pumps out 4 protons Complex 3 pumps out 4 protons Complex 4 pumps out 2 protons **Total of 10 protons per electron pair.** **NADH + 11 H⁺_M + ½ O₂ → NAD⁺ + 10 H⁺_IM + H₂O**

Answer 157

Proton pumping by ETC across the inner mitochondrial membrane sets up a pH gradient (ΔpH) and a transmembrane potential (Δψ) with matrix side negative. Can be calculated as ΔG = 2.3 RT ΔpH + F Δψ In general, transport of a pair of electrons through the ETC generates ~ 53 kcal.

Answer 158

The proton motive force drives ATP synthesis as the protons flow passively back through the inner mitochondrial matrix down their concentration gradient through a proton pore in the ATP synthase. The ~ 53 kcal produced per pair of electrons can drive the synthesis of 3 ATPs (using ~ 22 kcal) with the remaining energy used to drive ancillary reactions or dissipated as heat. ADP + P_i + *n*H⁺_IM → ATP + H₂O + nH⁺_M Value of *n* depends on the structure of the ATP synthase and varies between species (~3.3 - 5 protons per ATP)

Answer 159

**F₁F_o ATPase** O stands for oligomycin which blocks the proton pore F-type ATPase which functions as a reversible ATP-driven proton pump * F₁ portion contains the ATPase domain. * Contains 9 subunits α₃β₃γδε * α and β subunits for the knoblike catalytic section * γ subunit forms a shaft that connects with F_o * F_o complex consists of a, b, and c subunits * 8 c-subunits form the c-ring in vertebrates * Rotation of C-ring induces conformational changes in the β subunits of F₁ that drive ATP synthesis * Requires 8 protons moving across the membrane with each rotation and produces 3 ATP's per turn

Answer 160

* Takes **11 H⁺ to generate 3 ATP's** * 8 H⁺ to give a complete c-ring rotation * Additional 3 H⁺ to bring 3 phosphates into the matrix via the mitochondrial phosphate transporter * From 1 NADH: 10 H⁺ pumped so (10/11) x 3 ATPs produced = **2.75 ATPs per NADH** * From 1 FADH₂(Succinate): 6 H⁺ pumped so (6/11) x 3 ATPs produced = **1.64 ATPs per FADH₂** * Glycolysis generates 2 net ATPs and 2 NADH * PDH generates 1 NADH/pyruvate → 2 NADH/glucose * TCA generates: * 3 NADH/pyruvate → 6 NADH/glucose * 1 FADH₂/pyruvate → 2 FADH₂/glucose * 1 ATP/pyruvate → 2 ATP/glucose * OxPhos: * 10 NADH → 27.5 ATP * 2 FADH₂ → 3.3 ATP Going all the way through oxidative phosphorylation generates: * ~ 31 ATP by OxPhos * 2 ATP for glycolysis * 2 ATP for TCA **TOTAL OF ~35 ATP/GLUCOSE**

Answer 161

CN^- or CO blocks electron transport and subsequently ATP synthesis

Answer 162

Phosphorylation inhibitor Blocks ATP synthesis and thus electron transport

Answer 163

**dinitrophenol (DNP)** **uncoupling protein 1 (UCP1 aka thermogenin)** * Causes collapse of the proton motive force releasing energy in the form of heat rather than ATP. * Electrons free to move through the ETC. * Increases oxygen consumption.

Answer 164

ETC and ATP synthesis coupled so increasing or decreasing one does the same to the other.

Answer 165

All the transport processes associated with oxidative phosphorylation which utilize the proton motive force or transmembrane potential. Mostly used to transport molecules across the impermeable inner mitochondrial membrane. * Agents that disrupt the PMF will reduce transport. * Transport of these substances will conversely reduce the PMF available for ATP synthesis. * Electroneutral transport systems driven by concentration gradients alone. * Electrogenic transport systems utilize concentration gradients as well as the transmembrane potential. * positively charged molecules enter matrix more easily * Adenine nucleotide translocase * Phosphate translocase * Transporters for other charged metabolites such as pyruvate, malate, citrate, etc. * Ca⁺⁺ and asparate also transported in a PMF-dependent fashion

Answer 166

Antiporter that exchanges ADP^3- from the intermembrane space for ATP^4- from the matrix. Net transport of 1 negative charge out of the matrix (electrogenic) stimulated by the matrix-negative transmembrane potential. Inhibited by atractyloside.

Answer 167

Symporter that moves one phosphate (H₂PO₄^-) and one proton (H⁺) into the matrix. Driven by the proton gradient established by ETC. Electronically neutral.

Answer 168

Complex containing ATP synthase, adenine nucleotide translocase, and phosphate translocase can be isolated by gentle disruption of the inner mitochondrial membrane suggesting that these three proteins are spatially integrated.

Answer 169

A transmembrane protein embedded in the inner mitochondrial membrane. Uses the proton gradient to drive: **NADH + NADP⁺ + H⁺_IM → NAD⁺ + NADPH + H⁺_M** NADH is used by ETC so its levels are related to the potential for ROS generation. Production of NADPH by transhydrogenase will increase as more electrons travel down ETC. NADPH indirectly used in ROS neutralization.

Answer 170

Mitochondria contain their own genome which contains 37 genes. 13 encode subunits of respiratory chain proteins.

Answer 171

Maternally inherited. Defects in oxidative phosphorylation most commonly associated with mutations in mitochondrial genes. Presumably due to generation of reactive oxygen species. Affects tissues with high requirement for ATP such as brain, liver, and skeletal/cardiac muscle.

Answer 172

* Triggered by: * External signals acting via a receptor * Oxidative stress * Heat shock * Viral infection * Exposure to stimulus induces formation of large pores in the outer mitochondrial membrane called permeability transition complex * Allows release of cytochrome c into the cytosol * Cytochrome c in associated with apoptosis protease activating factor 1 (Apaf-1) activate a family of cytosolic proteases (the caspases) that degrade proteins and lead to cell death.

Answer 173

* Major short-term storage form of carbohydrates in animals. * For times of metabolic need. * Branched chain homopolysaccharide of α-D-glucose * Primary bond is α-1,4-glycosidic linkages. * Every 8-10 glucose residues there is a branch attached via a α-1,6-glycosidic linkage * Stored in the cytoplasm as large hydrated granules. * Each granule contains as many as 55,000 glucose units. * Found in the cytoplasm of liver and skeletal muscle cells primarily.

Answer 174

1. Diet 2. Degradation of glycogen 3. Gluconeogenesis

Answer 175

* Functions to maintain the blood glucose concentration particularly during the early stage of a fast. * Glucose rapidly released from liver glycogen. * Glucose able to enter systemic circulation due to presence of _glucose-6-phosphatase_ in the liver. * Hepatic glycogen stores ~24 hour supply of glucose. * Liver also able to synthesize glucose via _gluconeogenesis_.

Answer 176

* Serves as a fuel reserve for synthesis of ATP that will power muscle contraction. * Muscle glycogen is not available to other tissues because muscle lacks glucose-6-phosphatase.

Answer 177

1. Phosphorylation * Glycolysis utilizes glucose-6-phosphate. 2. Create a nucleotide sugar * Glycogenesis utilizes UDP-glucose. Alternate activation methods allows for both pathways to occur at the same time.

Answer 178

* Occurs in the cytoplasm and can be divided into two stages: 1. Chain synthesis 2. Chain branching I. Chain Synthesis **A. Synthesis of UDP-glucose.** 1. _Glucose_ is phosphorylated to _glucose-6-phosphate_. - By *glucokinase* in hepatic tissue. - By *hexokinase* in peripheral tissue. 2. _Glucose-6-phosphate_ converted to _glucose-1-phosphate_ by *phosphoglucomutase*. 3. _Glucose-1-phosphate_ reacts with _UTP_ to form _UDP-glucose_ and _PP_i_ which is catalyzed by *glucose 1-phosphate uridylyltransferase* (aka *UDP-Glc pyrophosphorylase*) - Hydrolysis of _PP_i_ → _2 P_i_ by *pyrophosphatase* makes the reaction energetically favorable and irreversible. **B. Requirement of primer to initiate glycogen synthesis.** ⇒Glycogen synthase cannot initiate glycogen synthesis de novo and can only add glucose to an existing chain, therefore, glycogenesis requires a primer. 1. _Glycogen_ _fragment_ can serve as a primer for *glycogen synthase* which attaches glucosyl residues to existing chain using _UDP-glucose_. 2. The protein *_glycogenin_* (homodimer) can prime glycogen synthesis and attach glucose residues through auto-glucosylation ⇒ serves as both substrate and enzyme in its role as primer. a. The *glycosyltransferase* activity of _glycogenin_ transfers the first molecules of glucose from _UDP-glucose_ to a specific _tyrosine side-chain_ (tyr-194) on itself. b. After at least 4 (about 7) glucose residues have been added, *glycogen synthase* takes over. - Glycogenin remains within the glycogen granule. **C. Elongation of glycogen chains.** 1. *Glycogen synthase* transfers glucose from _UDP-glucose_ to the _non-reducing end of the growing chain_ via α-1,4-linkages between the -OH group on C-1 of the activated sugar and the C-4 of the accepting sugar. ⇒ *Glycogen synthase* is the rate-limited and regulated enzyme of glycogenesis. ⇒ There are liver & muscle isozymes. ⇒ _UDP_ released when α-1,4-glycosidic bond is formed can be converted to _UTP_ by *nucleoside diphosphate kinase* + ATP.

Answer 179

* Occurs in the cytoplasm and can be divided into two stages: 1. Chain synthesis 2. Chain branching II. Chain branching A. Catalyzed by "branching enzyme" called *glucosyl 4:6 transferase.* 1. *Glucosyl 4:6 transferase* cleaves an _α-1,4-glycosidic bond_ from the non-reducing end of the glycogen chain producing a 6-8 glucosyl residues fragment. 2. Enzyme then transfers the fragment to another residue on the linear chain via an _α-1,6-glycosidic bond_. 3. Resulting new non-reducing end and old non-reducing ends can be further elongated by *glycogen synthase*. 4. After further elongation, new chains of 6-8 residues can be transferred to make additional branches.

Answer 180

1. Increases solubility of glycogen molecule. * Stored as hydrated granules. 2. Increases number of non-reducing ends to which new glucosyl residues can be added to or removed from glycogen. * Facilitates fast breakdown of glycogen into glucose when energy needed.

Answer 181

* Occurs primarily in the cytoplasm of liver and skeletal muscle cells. * Involves 2 stages: 1. Shortening of chains 2. Removal of branches

Answer 182

1. *Glycogen phosphorylase* uses **Pi** to cleave the α-1,4-glycosidic bonds between glucose residues at the non-reducing ends of the glycogen chains releasing _glucose-1-phosphate_. ⇒Enzyme is a homodimeric exoglucosidase ⇒Requires *pyridoxal phosphate (PLP)* coenzyme (derivative of Vit B6) ⇒Liver and muscle isozymes * *Glycogen phosphorylase* stops attacking α-1,4-glycosidic bonds _four glucosyl residues from an α-1,6-branch point_. * Resulting structure is called a **limit dextrin.** 2. ****_Glucose-1-phosphate_ is converted to _glucose-6-phosphate_ by *phosphoglucomutase*. 3. Next step in glycogenolysis depends on the tissue: * In liver: _glucose-6-phosphate_ is hydrolyzed to _free glucose and P_i_ by *glucose-6-phosphatase*. ⇒Free glucose able to leave the liver and enter the blood stream. * In peripheral tissues: glucose-6-phosphate will be oxidized in the glycolytic pathway to produce energy.

Answer 183

Catalyzed by a debranching enzyme which is a bifunctional protein with two catalytic activities. 1. *4-α-D-glucantransferase* activity transfers the _outer three glucosyl residues_ of the _limit dextrin_ to a _non-reducing end_ by breaking and formation of α-1,4 bonds leaving one glucosyl residue in α-1,6 linkage. ⇒ 4,4 transferase activity 2. _α-1,6 linkage_ is then cleaved hydroytically by the *amylo-α-1,6 glucosidase* activity of debranching enzyme releasing _free glucose_ (non-phosphorylated). 3. Co-operative and repetitive action of phosphorylase and debranching enzymes results in complete hydrolysis of glycogen to yield glucose-1-phosphate and free glucose in a 12:1 ratio. ⇒Free glucose is quickly phosphorylated in muscle for intracellular use. ⇒Phosphorylated glucose is already trapped inside muscle cells.

Answer 184

_Key regulatory enzymes of glycogen metabolism:_ Glycogen synthase (synthesis) & Glycogen phosphorylase (degradation) Each is controlled by: 1. _Hormone-induced covalent modification_ through phosphorylation or dephosphorylation of ser residues. * Way of responding to the needs of the body as a whole. 2. _Allosteric effectors_ * Way of responding to the need of a particular tissue at a particular time.

Answer 185

**I. Regulation by covalent modification:** Glycogen phosphorylase exists in two forms: _A_ or active form: phosphorylated _B_ or inactive form: dephosphorylated A and B forms are interconverted by: *Phosphorylase kinase* Produces active phosphorylated A-form. & *Phosphoprotein phosphatase-1* Produces inactive dephosphorylated B-form. \*The A form of glycogen phosphorylase is more active because phosphorylation causes a conformational change in the enzyme which shifts the equilibrium of the enzyme between its T-state (taut and inactive) and R-state (relaxed and active) towards the active R-state. The B-form is inactive because the taut state is favored. *Phosphorylase kinase* (regulatory enzyme) is itself regulated by phosphorylation/dephosphorylation. A and B forms are interconverted by: *Protein Kinase A (PKA)* Produces active phosphorylated A-form. & *Phosphoprotein phosphatase-1* Produces inactive dephosphorylated B-form.

Answer 186

Hormonal signals that activate PKA include Glucagon and Epinephrine. Phosphorylase kinase (regulatory enzyme) & Glycogen phosphorylase (regulated enzyme of glycogenolysis) are phosphorylated in response to hormonal signals that are transduced via cAMP which activates protein kinase A. PKA also results in the **inhibition** of *phosphoprotein phosphatase-1* by phosphorylating and activating *protein phosphatase inhibitor* (A-form). This enzyme is used to maintain the level of phosphorylation until hormone signal changes. Active protein kinase A has a short half-life because seperation of regulatory and catalytic subunits revealed PEST sequences which marks protein for degradation.

Answer 187

Peptide hormone released from the alpha-cells of the pancreas when blood glucose is low. Glucagon binds to plasma membrane receptors on liver cells but not muscle. Stimulates glycogen degradation via PKA-mediated activation of phosphorylase kinase. Activated during periods of fasting thus making glucose available to tissues.

Answer 188

*Glycogen synthase* exists in two forms: A or active form: dephosphorylated B or inactive form: phosphorylated Several kinases phosphorylate glycogen synthase A to the inactive B form including: * cAMP dependent PKA* * Phosphorylase kinase* * Glycogen synthase kinase-3* * AMP-dependent kinase* B-form converted back into the active A-form by *Phosphoprotein phosphatase-1.* PKA also results in the inhibition of phosphoprotein phosphatase-1 by phosphorylating and activating *protein phosphatase inhibitor* (A-form).

Answer 189

cAMP regulates glycogen metabolism through the simultaneous activation of glycogenolysis and inhibition of glycogenesis. Also displays amplification: 1 hormone activates many adenyl cyclase, makes many cAMP, each activates many PKA, each phosphorylates many phosphorylase kinase, each phosphorylates many phosphorylase ect.

Answer 190

Released by pancreas in response to high blood glucose levels. Has the opposite affect of glucagon/epinephrine. Activates glycogen synthesis and inhibits degradation in liver and muscle. 1. Promotes inhibition of several protein kinases and activation of phosphoprotein phosphatase. * Causes subsequent dephosphorylation of glycogen synthase (activating) and dephosphorylation of phosphorylase kinase and phosphorylase (inactivating). 2. Promotes conersion of cAMP to 5' AMP by activating a phosphodiesterase. * Causes subsequent decrease in active PKA.

Answer 191

* Allosteric effectors are superimposed onto covalent regulation in order to meet the needs of the tissue. * Enzymes of glycogen metabolism including glycogen phosphorylase kinase, glycogen phosphorylase, and glycogen synthase are regulated in an allosteric effectors acting in a _non-covalent manner_. * Postive allosteric effectors bind to a regulatory site on the R (active) form of the enzyme stabilizing it and pulling the equilibrium towards the R-form. * Negative allosteric effectors bind to the T (inactive) form and stabilizes that pulling equilibrium towards T-form. * Effectors include: * Ca²⁺ and AMP which are signs of low energy * Glucose and glucose-6-phosphate which are signs of high energy

Answer 192

Released in times of energy need. Ca²⁺ binds to the calmodulin-like δ-subunits of dephosphorylated (B-form) *phosphorylase kinase* causing conformational change which activates the catalytic γ-subunits in the absence of phosphorylation ⇒ stabilizes the R-state Ca²⁺ also required for maximal activation of phosphorylase kinase a. *Phosphorylase kinase* subsequently phosphorylates and inhibits *glycogen synthase*. End result of a rise in [Ca²⁺]_in is increased degradation and decreased synthesis of glycogen. * **Contracting muscle** * Ca²⁺ released in in response to nerve impulses. * [Ca²⁺]_in activates sarcoplasmic glycogenolysis by activating phosphorylase kinase. * **Liver cells** * Epinephrine binds to α-adrenergic receptors, activating phospholipase-C, and generating IP₃ and DAG from PIP₂. * IP₃ causes release of Ca²⁺ from the SER. * [Ca²⁺]_in activates glycogenolysis by activating phosphorylase kinase⇒ activates glycogen degradation. * DAG activates PKC which phosphorylates and inactivates glycogen synthase ⇒ inhibites glycogen synthesis. Side note: Ca²⁺ also activates mitochondrial events: * Activates PDH phosphatase thereby activating PDH and pyruvate degradation. * Allosteric effector of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase thereby activating TCA cycle.

Answer 193

* AMP to ATP ratio reflects the energy state in muscle cells. * Increase in AMP signals low energy and need for glycogen degradation. * AMP functions as an allosteric activator of muscle phosphorylase b. * Directly activates the dephosphorylated form of myophosphorylase b.

Answer 194

* When glucose is plentiful, _heptatic_ glycogenolysis is decreased by glucose itself acting as an allosteric inhibitor of hepatic phosphorylase a. * The glucose-6-phosphate fromed from glucose is an allosteric activator of glycogen synthase b in _both liver and muscle_, thus increasing glycogenesis. * For this reason, glycogen synthase b is sometimes designated "D" because it is dependent on glucose-6-phosphate for activity while synthase a is designed "I" for independent.

Answer 195

**Von Gierke Disease** * **Glucose-6-phosphatase deficiency** * Liver and kidney * Severe fasting hypoglycemia hallmark * Major Findings * Hepato/renomegaly * Fasting hypoglycemia * Lactic acidemia * Hyperuricemia * Hyperlipidemia

Answer 196

**Pompe Disease** aka Generalized Glycogenosis * **Lysosomal acid α-glucosidase deficiency** * Infantile, juvenile, and adult-onset forms * Affects all organs but skeletal/cardiac most * Cardiomegaly/myopathy in infantile forms * Muscle weakness in later forms * Enzyme replacement therapy has reduced mortality

Answer 197

**Cori Disease** aka Limit Dextrinosis * **Debranching enzyme deficiency** (both actions) * IIIa : affects liver and muscle * IIIb: affects liver only * Milder hepatomegaly * Muscle weakness * Accumulated glycogen has abnormal structure with shorter chains * May cause liver fibrosis or cirrhosis

Answer 198

**Andersen Disease** aka Amylopectinosis * **Branching enzyme deficiency** in liver * Progressive hepatomegaly * Accumulated glycogen has abnormal structure with longer chains and no branches * Progressive liver cirrhosis in infantile form can be lethal

Answer 199

**McArdle Disease** * **Muscle glycogen phosphorylase deficiency** * Infantile and Adult forms * Exercise intolerance and muscle cramps * Symptoms usually first appear in adolescence * _Failure of blood lactate to rise_ after anaerobic exercise

Answer 200

**Hers disease** * **Liver glycogen phosphorylase deficiency** * Hepatomegaly * Benign in general * Mild fasting hypoglycemia

Answer 201

**Tarui Disease** * **Muscle PFK-1 deficiency** * Affects muscle and RBC's * More severe exercise intolerance and muscle cramps * RBC's show some percentage of normal activity

Answer 202

* The synthesis of glucose from smaller precursors such as: * pyruvate * lactate * glycerol * most amino acids * Alanine ⇒ pyruvate * Glutamine ⇒ α-ketoglutarate * Except: leucine and lysine * Serves to stabilize glucose levels in the blood during fasting after glycogen stores have been depleted * Occurs in the liver and to some extent the kidneys * Requires specialized gluconeogenic enzymes to bypass the irreversible steps of glycolysis

Answer 203

1. _Pyruvate + HCO₃^- → oxaloacetate_ by pyruvate carboxylase + ATP Reaction is *anaplerotic* because it yields a citric acid cycle intermediate OAA. 2. _Oxaloacetate → PEP + CO₂_ by phosphoenolpyruvate carboxykinase (PEPCK) + GTP Required to bypass the highly exergonic reaction catalyzed by pyruvate kinase during glycolysis.

Answer 204

* Biotin-containing multifunctional protein * Biotin coenzyme linked to a lysine residue During course of reaction: 1. Carbon dioxide is first bound to the biotin in one active site forming a high energy carboxy-biotin species. 2. Long flexible side chain swings the carboxy-biotin to the second active site. 3. Carboxy-group transferred to pyruvate.

Answer 205

_Allosterically activated by acetyl CoA._ Acetyl CoA is a product of fatty acid metabolism. Links gluconeogenesis and fat catabolism. Acetyl CoA also inhibits pyruvate dehydrogenase by activating PDH kinase. Decreased glycolysis, increased gluconeogensis, and increased fatty acid catabolism by the liver when blood glucose low. Compartmentalized in the mitochrondria.

Answer 206

_Fructose-1,6-bisphosphate_ → _fructose-6-phosphate_ by *fructose-1,6-bisphosphatase* By passes the reaction of PFK1 in glycolysis. fructose-1,6-bisphosphatase Allosterically inhibited by F-2,6-P₂ and AMP Same allosteric activators of PFK1 = Reciprocal regulation

Answer 207

_Glucose-6-phosphate_ → _glucose_ by *glucose-6-phosphatase* By passes reaction of hexokinase/glucokinase. Allows glucose to leave the cell and enter the blood.

Answer 208

Glucose-6-Phosphatase is a membrane bound enzyme located in the ER. Active site faces into the lumen of ER. Glucose-6-P must enter then glucose and P_i must leave. Deficiency causes glycogen storage disease Ia. Found only in liver and kidney. There is no direct control of the enzyme but has a high KM for glucose-6-P so only functions when concentrations high.

Answer 209

Glycolysis converts 1 glucose into 2 molecules of pyruvate. Produces 2 NADH and 2 ATP Gluconeogenesis converts 2 pyruvates into 1 glucose Consumes 2 NADH and 6 ATP or GTP Two processes are tightly regulated so that only once may proceed at a time. **_Allosteric Regulation_** Important sites for control are the irreversible reactions * pyruvate kinase - F-1,6-P₂ (+) , ATP (-) pyruvate carboxylase - Acetyl CoA (+), ADP (-) * phosphofructokinase - AMP (+) , F-2,6-P₂ (-) fructose bisphosphatase - AMP (-) , F-2,6-P₂ (-) **_Hormonal Regulation_** * Glucagon → increased cAMP → phosphorylation of hepatic pyruvate kinase and PFK 2 → decrease glycolysis * Insulin → decreased cAMP → increase hepatic glycolysis **_Transcriptional Regulation_** * Glucagon → stimulates expression of PEPCK and maybe glucose-6-phosphatase through phosphorylation of cAMP response element (CREB) by PKA * Insulin → stimulates expression of PFK1, pyruvate kinase, PFK2, enzymes of glycolysis

Answer 210

Gluconeogenesis requires both mitochrondrial and cytosolic enzymes. Pyruvate carboxylase is a mitochondrial enzyme while the other gluconeogenic enzymes are largely cytoplasmic. Pyruvate must be transported into the mitochondria where it is converted to OAA. OAA_mito ⇒ Malate_mito ⇒ Malate_cyto ⇒ OAA_cyto Gluconeogenesis consumes NADH in the cytosol in the conversion of 1,3-bisphosphoglycerate to glyceraldehyde-3-P but the NADH/NAD⁺ ratio is normally very low so glycolysis usually preferred.

Answer 211

* Occurs in the liver * _Lactate_ converted to _pyruvate_ in the cytosol by *LDH* * Yields NADH ⇒ export of reducing equivalents fro mthe mitochondria is not nescessary * Pyruvate enters mitochondria where it is converted to _OAA_ by *pyruvate carboxylase* * OAA can either: * Be converted to _aspartate_ by transamination in mitochondria ⇒ leave mitochondria via aspartate shuttle ⇒ converted back to _OAA_ in the cytosol * Be converted to _PEP_ by mitochrondrial *PEP carboxykinase* ⇒ PEP leaves mitochondria

Answer 212

Recycles glucose carbons from lactate in order to maintain blood glucose levels. Lacate produced via anaerobic glycolysis travels to the liver. There it is used for gluconeogenesis and resulting glucose is released back in to the blood. (Liver hopes the next time the glucose will be used oxidatively for more energy.)

Answer 213

Hypoxanthine

Answer 214

Orotic Acid

Answer 215

Built on a ribose-5-phosphate. Occurs primarily in cytosol of hepatocytes.

Answer 216

**11 step process** Requires a large amount of ATP 1. **Ribose-5-P ⇒ PRPP** by ***PRPP synthetase*** * _Regulated_ but not committed step 2. Add **N from glutamine** to PRPP by ***PRPP glutamyl amidotransferase*** * Committed step * Highly regulated * Rate-limiting 3. Add **glycine backbone** 4. Add **C from N¹⁰-THF** 5. Add **N from glycine** for ring 2 6. **Ring 1 closure** 7. Add **C from respiratory CO₂** 8. Attach **aspartate R group N** to C of respiratory CO₂ 9. Keep only N from aspartate and release backbone as fumarate 10. Add **C from N¹⁰-THF** 11. **Close ring 2** to form **inosine monophosphate (IMP)**

Answer 217

Catalyzes the **regulated but not committed step** of purine de novo synthesis. _Feed back inhibition_ by purine ribonucleotides (mainly **AMP and GMP)** _Activated_ by **P_i**

Answer 218

Catalyzed the committed and rate-limiting step of purine de novo synthesis. _Inhibited_ by **end-products** (purine ribonucleotides, inactive dimer) _Activated_ by **PRPP** and active monomer **High [PRPP] able to overcome end-product inhibition.**

Answer 219

IMP converted to AMP or GMP in seperate two step reactions. **_IMP ⇒ AMP_** IMP + Asp ⇒ Adenylosuccinate monophosphate (ASMP) ASMP ⇒ AMP + Fumarate **_IMP ⇒ GMP_** IMP ⇒ XMP XMP + Gln ⇒ GMP + Glutamate

Answer 220

**Reciprocal regulation** ensures appropriate levels of AMP and GMP. 1st step in IMP ⇒ AMP inhibited by AMP and requires GTP. 2nd step in conversion of IMP ⇒ GMP inhibited by GMP and requires ATP.

Answer 221

Meets the needs of the cell.

Answer 222

Maintains adequate and balanced concentrations of deoxyribonucleotides. Catalyzed by ***ribonucleotide reductase***. Requires reduced **thioredoxin** coenzyme. _RNR has 3 sites:_ * **Active site** ⇒ reduces ribonucleotide to deoxyribonucleotide * **Activity site** ⇒ _on/off switch_ * ATP activates * dATP inactivates * **Substrate specificity site** ⇒ binds a _specific positive allosteric effector_ (NTP or dNTP) to control reductive of a given NDP * C ⇒ U ⇒ G ⇒ A * Ex. Binding of dCTP makes enzyme specific for UDP RNR inhibited pharmacologically by **hydroxyurea**.

Answer 223

Guanine ⇒ Xanthine by Guanase Adenosine ⇒ Inosine by Adenosine Deaminase Inosine ⇒ Hypoxanthine by Purine nucleoside phosphorylase Hypoxanthine ⇒ Xanthine by Xanthine Oxidase Xanthine ⇒ Uric acid by Xanthine Oxidase

Answer 224

Free purine bases or nucleosides can be reutilized. Important in non-hepatic tissues. **_Base salvage:_** ***Adenine phosphoribosyltransferase (APRT)*** Adenine + PRPP ⇒ AMP + PP_I ***Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)*** H or G + PRPP ⇒ IMP or GMP + PP_i **_Nucleoside salvage:_** ***Adenosine kinase*** Adenosine + P_i ⇒ AMP

Answer 225

**_Salvage inhibits de novo synthesis:_** **Decreases [PRPP]** ⇒ base salvage only **Generates nucleoside monophosphates that inhibit the *amidotransferase*** ⇒ base and nucleoside salvage

Answer 226

Adenosine Deaminase (ADA) Deficiency ⊗ ADA ⇒ ⊗ adenosine catabolism ⇒ ↑ [dATP] ⇒ ⊗ RNR ⇒ developmental arrest and/or apoptosis of lymphocytes Results in severe combined immunodeficiency syndrome ⇒ **ADA-SCIDS**

Answer 227

Purine nucleoside phosphorylase deficiency Rarer and less severe than ADA deficiency. **Primarily affects T-cells.**

Answer 228

* X-linked * Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT) deficiency * ↑ [PRPP] ⇒ ↑ [uric acid] * Symptoms: * hyeruricemia with gout * compulsive self-mutilation * aggressive behavior * developmental delay * profound motor impairment * early death from renal failure

Answer 229

Build the base directly then attach to a sugar phosphate.

Answer 230

Pyrimidine ring made then linked to ribose phosphate. CAD catalyzed first 3 reactions. UMP synthase catalyzes reactions 5 & 6. * **Orotic acid** is the first pyrimidine formed * **Regulated and rate-limiting step** is synthesis of carbamoyl phosphate by *CPS II.* * **Committed step** is synthesis of _carbamoyl aspartate_ by *aspartate transcarbamoylase.* * Orotic acid + PRPP ⇒ OMP (first nucleotide) * OMP decarboxylated to UMP

Answer 231

OMP ⇒ UMP * UMP phosphorylated by kinases to UDP and UTP * **UTP** _aminated_ using **Gln** to **CTP** and Glu by ***CTP synthetase***

Answer 232

⊕ UTP ⊖ CTP

Answer 233

**UDP ⇒ dUDP** by *RNR* **_dUMP arises from both dUD(T)P and dCMP_** dUDP ⇒ dUTP by kinase dUTP ⇒ dUDP ⇒ dUMP by **dUTPase** dCMP ⇒ dUMP by deamination

Answer 234

**dUMP** _methylated_ at the 5 position by ***thymidylate synthase*** ⇒ **TMP** Thymidylate synthase inhibited by **5FU**. Uses **N⁵N¹⁰-methylene THF** as 1 C donor. THF oxidized to DHF as methylene group reduced to methyl group. DHF reduced back to THF by ***dihydrofolate reductase* + NADPH**. Dihydroflocate reductase inhibited by **methotrexate**. Traps folate as DHF ⇒ ↓ thymidylate synthesis ⇒ ↓ DNA synthesis

Answer 235

5-FU converted to nucleotide monophosphate by addition of PRPP. Deoxy from of nucleotide **inhibits thymidylate synthase.**

Answer 236

Folic acid analog **Inhibits dihydrofolate reductase.** ↓ thymidylate synthesis ⇒ ↓ DNA synthesis Used to treat some cancers and psoriasis.

Answer 237

Trifunctional protein. Catalyzes the first 3 steps of pyrimidine synthesis: 1. Carbamoyl phosphate synthetase II 2. Aspartate transcarbamoylase 3. Dihyrorotase

Answer 238

Carbamoyl phosphate synthetase II is the regulated enzyme ⊕ PRPP ⊖ UTP

Answer 239

**UMP synthase deficient** 1. Orotate phosphoribosyl transferase 2. Orotate decarboxylase ↑ [orotic acid] and ↑ excretion of OA Symptoms are failure to grow and develop and anemia. **Treat with uridine:** Uridine → UMP →→ UTP → CTP Requires functional salvage pathway. UTP inhibits CPSII of pyrimidine de novo synthesis so decreases production of OA also.

Answer 240

All products are water soluble. There are no known pathologies of salvage or degradation. Nucleotide → Nucleoside → Free base: **Cytosine** _deaminated_ to **uracil**. **Uracil** → β-alamine, CO₂ and NH₄ **Thymine** → β-aminoisobutyrate, CO₂ and NH⁺ **Some salvage** of nucleosides and bases to nucleotides.

Answer 241

Thymine → β-aminoisobutyrate, CO₂ and NH⁺ β-aminoisobutyrate is unique to thymine degradation and urinary excretion **used as a measure of DNA turnover**

Answer 242

Disorder of purine metabolism. Requires: Hyperuricemia Precipitation of uric acid or monosodium urate crystals in tissues Inflammatory response Tophi may be seen.

Answer 243

Uric acid deprotinated at physiological pH to urate Urate + sodium ions ⇒ monosodium urate

Answer 244

1. **Hyperuricemia** * Necessary precondition for gout but not enough * All patients with gout show hyperuricemia but only minority of people with hyperuricemia experience gout 2. **Formation and deposition of urate crystals** * Tendency for crystal deposition varies * Factors may precipitate crystal formation * ↓ temperature * Explain why gout most commonly affects great toe (podagra) * ∆ pH * trauma * stress 3. **Inflammatory response to the crystals**

Answer 245

Asymptomatic hyperuricemia ↓ Acute intermittent attacks ↓ Chronic tophaccous gout May see urolithiasis.

Answer 246

**Gout is the major manisfestation of an innate disorder.** \>90% of cases hyperuricemia due to ↓ excretion not ↑ production Most cases idiopathic. Known molecular defects: * PRPP synthetase with increased activity ⇒ super synthetase * Partial deficiency of HGPRT

Answer 247

Gout is a secondary manifestation of an underlying disorder. * Total deficiency (\<1.5% of normal) of HGPRT seen in X-linked Lesch-Nyhan syndrome. * Increased cell turnover * cancers * hemolytic diseases * psoriasis * chemotherapy * Decreased renal excretion * lead poisoning * Increased conversion of ATP ⇒ AMP * ETOH abuse * Type I glycogen storage disease * Inborn errors of fructose metabolism * Hypoxia

Answer 248

Therapy to reduce inflammation. No effect on urate concentrations. * **NSAIDS** * **Indomethacin** * Aspirin contraindicated * competes with urate for renal excretion * **Colchicine** * Inhibits phagocytosis of urate crystals * Inhibits microtubule assembly * Many side effects * Low dose used prophylactically * **Steroids** * Glucocorticoids

Answer 249

Life-long therapy to decrease urate concentration. No direct effect on inflammation. * Increasing renal excretion ⇒ **uricosuric agents** * **Probenecid** * Inhibits renal reabsorption of urate * Decreasing urate synthesis * **Xanthine oxidase inhibitors** * **Allopurinol** * **Febuxostat** * **Uricase** * urate to more soluble allantoin

Exam 2: Biochemistry Flashcards

(286 cards)