EXAM II review Comprehensive Biochemistry Review: Metabolism, Glycosylation, and Bioenergetics

Principles of Bioenergetics and Metabolic Regulation

• Standard free energy changes ($\Delta G^{\circ\prime}$) serve as reference values, but actual free energy depends on substrate and product concentrations; an endergonic reaction ($\Delta G^{\circ\prime} > 0$) can proceed forward if coupled to a sufficiently exergonic reaction ($\Delta G^{\circ\prime} < 0$).

• Hydrolysis of $ATP$ to $ADP$ releases $-30\,\text{kJ\,mol}^{-1}$; hydrolysis to $AMP$ releases pyrophosphate, which is cleaved into two inorganic phosphates ($P_i$) to release additional free energy.

• Oxidation of $NADH$ releases a substantial amount of negative free energy, which is coupled to synthesize $ATP$ from $ADP$ and $P_i$.

• Coordinated metabolic control follows a universal trend: high-energy states ($ATP$, $NADH$, acetyl CoA) inhibit catabolic pathways, whereas low-energy states ($ADP$, $AMP$, $NAD^+$, CoA) activate them.

• Regulatory mechanisms include allosteric modulation, substrate availability, enzyme concentration, and covalent modifications (phosphorylation typically inhibits catabolic enzymes, while dephosphorylation activates them).

Carbohydrate Structure and Protein Glycosylation

• Carbohydrate isomers ($CH_2O$):

  • Constitutional isomers: Differ in atomic bonding connectivity (e.g., dihydroxyacetone vs. glyceraldehyde).

  • Stereoisomers: Include enantiomers (non-superimposable mirror images), diastereoisomers (non-mirror-image stereoisomers), epimers (differ at only one asymmetric carbon), and anomers (differ at the stereocenter generated during ring closure).

• N-linked glycosylation:

  • Occurs cotranslationally on the side chain of asparagine (Asn) within the endoplasmic reticulum (ER).

  • An oligosaccharide is built unit-by-unit on a dolichol phosphate lipid anchor, transferred to the nascent polypeptide by oligosaccharide transferase, and is required for proper protein folding.

  • Transport vesicles move the glycoprotein from the cis to trans Golgi for further carbohydrate remodeling.

  • Proteins targeted to lysosomes receive a mannose 6-phosphate tag in the Golgi via phosphoryl transferase and phosphodiesterase, which is recognized by the mannose 6-phosphate receptor.

• O-linked glycosylation:

  • Occurs entirely post-translationally in the Golgi apparatus.

  • Polypeptide N-acetylgalactosaminyltransferase attaches UDP-GalNAc to the hydroxyl side chain of serine (Ser) or threonine (Thr).

Glycolysis Pathway and Regulation

• Preparatory phase (first 5 enzymes):

  • Consumes $2\,\text{ATP}$.

  • Hexokinase phosphorylates glucose to prevent exit through glucose transporters; phosphoglucose isomerase yields fructose 6-phosphate; phosphofructokinase (PFK) adds the second phosphate; aldolase splits the hexose into two 3-carbon sugars; triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP).

• Payoff phase (next 5 enzymes):

  • Repeated twice per glucose molecule to yield a net of $2\,\text{ATP}$ and $2\,\text{NADH}$.

  • Glyceraldehyde 3-phosphate dehydrogenase carries out the initial oxidation-reduction reaction to generate $NADH$ and 1,3-bisphosphoglycerate.

  • Phosphoglycerate kinase and pyruvate kinase transfer high-energy phosphate groups to $ADP$ via substrate-level phosphorylation.

  • Phosphoglycerate mutase relocates the phosphate group, and enolase dehydrates the substrate to form phosphoenolpyruvate (PEP).

• Regeneration of cytosolic $NAD^+$ is essential for glycolytic flux; accomplished anaerobically via lactate fermentation or aerobically through mitochondrial shuttles (glycerol 3-phosphate shuttle or malate-aspartate shuttle).

• Regulation of irreversible glycolytic enzymes:

  • Hexokinase: Inhibited by feedback accumulation of glucose 6-phosphate.

  • Phosphofructokinase-1 (PFK-1): Allosterically inhibited by $ATP$, citrate, and protons ($H^+$); allosterically activated by $AMP$ and fructose 2,6-bisphosphate (synthesized by phosphofructokinase-2).

  • Pyruvate kinase: Activated by feed-forward interaction with fructose 1,6-bisphosphate; inhibited by $ATP$ and alanine. Regulated hormonally by phosphorylation (glucagon induces protein kinase to inactivate it; insulin activates a phosphatase to dephosphorylate and activate it).

Pyruvate Dehydrogenase Complex and Citric Acid Cycle

• Pyruvate Dehydrogenase Complex (PDC):

  • Converts pyruvate, $CoA$, and $NAD^+$ into acetyl CoA, $CO_2$, and $NADH$.

  • Enzyme 1 (pyruvate dehydrogenase): Catalyzes oxidative decarboxylation using thiamine pyrophosphate (TPP) cofactor.

  • Enzyme 2 (dihydrolipoyl transacetylase): Transfers the acetyl group to CoA via a lipoamide cofactor.

  • Enzyme 3 (dihydrolipoyl dehydrogenase): Re-oxidizes dihydrolipoamide using $FAD$ and $NAD^+$.

  • Regulated allosterically and covalently: inhibited by phosphorylation, $ATP$, $NADH$, and acetyl CoA; activated by dephosphorylation, $ADP$, and pyruvate.

• Citric Acid Cycle net yield:

  • Each acetyl CoA oxidized releases $2\,\text{CO}_2$, $3\,\text{NADH}$, $1\,\text{FADH}_2$, and $1\,\text{GTP}$ (or $ATP$).

  • Regulated by energy status: inhibited by $ATP$, $NADH$, and succinyl CoA; activated by $ADP$, $AMP$, $NAD^+$, and CoA.

Electron Transport Chain and ATP Synthesis

• Redox potential:

  • A lower or more negative reduction potential designates a stronger reductant with higher electron transfer potential.

  • Electrons flow sequentially from negative to positive redox potential: $NADH \rightarrow \text{Complex I} \rightarrow \text{ubiquinone} \rightarrow \text{Complex III} \rightarrow \text{cytochrome } c \rightarrow \text{Complex IV} \rightarrow O_2$.

• Electron carriers:

  • $NADH$ and $FADH_2$ transfer pairs of electrons.

  • Ubiquinone (Q) accepts two electrons one at a time via a stable semiquinone radical intermediate.

  • Cytochrome c accepts and transfers single electrons ($1\,e^-$).

• Proton translocation across the inner mitochondrial membrane:

  • Complex I pumps $4\,H^+$ from the matrix to the intermembrane space.

  • Complex II (succinate dehydrogenase) transfers electrons directly from succinate to ubiquinone without pumping protons.

  • Complex III pumps protons via the Q cycle.

  • Complex IV pumps $4\,H^+$ per 4 electrons reduced, while chemically consuming $4\,H^+$ from the matrix to reduce $O_2$ into $2\,\text{H}_2\text{O}$.

• ATP Synthase structure and mechanism:

  • Proton-conducting unit ($F_0$): Consists of the stationary a subunit (containing two half-channels) and the rotating c-ring.

  • Rotating central stalk: The $\gamma$ subunit rotates alongside the c-ring.

  • Catalytic unit: The stationary $\alpha_3\beta_3$ hexamer is held rigid by the peripheral stalk/exterior column (composed of the b dimer and $\delta$ subunit).

  • Binding change mechanism: As the $\gamma$ subunit turns, the $\beta$ catalytic subunits cycle through three conformations: Open (releases $ATP$, binds $ADP$ and $P_i$), Loose (traps $ADP$ and $P_i$), and Tight (catalyzes the synthesis of $ATP$).

Shuttles and Respiratory Control

• Glycerol 3-Phosphate Shuttle:

  • Cytosolic glycerol 3-phosphate dehydrogenase oxidizes $NADH$ to $NAD^+$ while reducing dihydroxyacetone phosphate to glycerol 3-phosphate.

  • Mitochondrial glycerol 3-phosphate dehydrogenase re-oxidizes the substrate using enzyme-bound $FAD$, passing electrons to ubiquinone.

  • Bypasses Complex I, causing cytosolic $NADH$ using this shuttle to yield only $2\,\text{ATP}$.

• Adenine Nucleotide Translocase (ATP-ADP Translocase):

  • An antiporter in the inner mitochondrial membrane that imports one cytosolic $ADP$ into the matrix in exchange for exporting one matrix $ATP$.

• Respiratory control:

  • The rate of oxidative phosphorylation is governed strictly by the availability of cytosolic $ADP$.

  • When cytosolic $ATP$ is high and $ADP$ is low, the translocase limits $ADP$ entry into the matrix, halting ATP synthase activity.

  • The resulting elevated proton gradient stops the electron transport chain, causing $NADH$ to accumulate, which allosterically inhibits the citric acid cycle and PDC.