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Biochemistry - 8 Open Questions: Full Exam Guide

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1. Biosynthesis of Catecholamines: Steps & Enzymes

Step-by-Step Pathway

Key Points for Exam

• Tyrosine hydroxylase is the rate-limiting step - it is a mixed-function oxidase regulated by end-product (catecholamine) feedback inhibition. Catecholamines compete with BH4 for the enzyme's cofactor-binding site. Depolarization activates kinases (PKA, PKC, CaM kinases) that phosphorylate TH, making it less sensitive to feedback.
• Step 2 (AADC) uses PLP and is the same enzyme that decarboxylates 5-HTP to serotonin. Dopaminergic neurons stop at dopamine - they do not have DBH or PNMT.
• Step 3 (DBH) occurs exclusively inside storage vesicles (not in the cytosol), requires vitamin C (ascorbic acid) as electron donor, and Cu2+ as a bound cofactor. Disulfiram (Antabuse) inhibits DBH.
• Step 4 (PNMT) depends on SAM for methyl donation, making it dependent on adequate vitamin B12 and folate (both needed to regenerate SAM from homocysteine).
• L-DOPA is the principal drug for Parkinson's disease - it bypasses the rate-limiting TH step and is converted to dopamine by AADC in the brain.

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2. Mechanism of Histamine Action + Food Poisoning Cases

Biosynthesis & Release

Four Receptor Types - Mechanisms & Effects

Cardiovascular & Other Actions

• Vasodilation of resistance vessels (both H1 and H2) → lowers blood pressure; increases capillary permeability → edema formation
• Bronchoconstriction via H1 in lungs (key in anaphylaxis)
• Sensory nerves - stimulates itching via H1

Inactivation of Histamine

• In the brain: Histamine methyltransferase (HNMT) methylates histamine using SAM → N-methylhistamine → then oxidized by MAO-B
• In peripheral tissues: Diamine oxidase (DAO) deaminates histamine → then oxidized to a carboxylic acid
• Note: there is no reuptake transporter for histamine (unlike monoamines)

Food Poisoning Cases Involving Histamine

• Occurs when poorly refrigerated dark-flesh fish (tuna, mackerel, mahi-mahi, sardines) decompose. Bacteria (Morganella morganii, Klebsiella pneumoniae) decarboxylate histidine to histamine via bacterial histidine decarboxylase.
• Symptoms within minutes to 1-2 hours: flushing, rash/urticaria, nausea, vomiting, diarrhea, headache, palpitations, burning sensation in the mouth, bronchoconstriction.
• Mimics an allergic reaction but is a direct pharmacological effect of excessive histamine absorption.
• Treatment: antihistamines (H1 + H2 blockers).

• Improperly fermented cheeses, wine, beer, and sauerkraut can accumulate high histamine levels.
• In susceptible individuals with low intestinal diamine oxidase (DAO) activity, dietary histamine is not degraded and enters systemic circulation.
• Symptoms: headache, flushing, GI distress, palpitations.

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3. Compounds that Inactivate Peptide Neurotransmitters

Main Classes of Inactivating Enzymes

Key Characteristics of Peptide NT Inactivation

1. No reuptake - unlike dopamine, serotonin, GABA (which have specific plasma membrane transporters), peptides have no known reuptake mechanism after release.
2. Enzymatic cleavage is irreversible - the signal ends when the peptide is degraded.
3. Enzymes are ectoenzymes - located on the outer surface of the plasma membrane (e.g., neprilysin on postsynaptic neurons, glia, and endothelial cells), enabling them to act at the synapse or at some distance (volume transmission).
4. Diffusion also contributes - peptides diffuse away from the release site and are eventually cleared.
5. Different proteases for different peptides - each neuropeptide's inactivation involves a specific set of enzymes depending on its structure.

Clinical Relevance

• Enkephalinase (neprilysin) inhibitors (e.g., sacubitril in combination with valsartan = Entresto) were originally developed to reduce enkephalin degradation for analgesia; sacubitril is now used in heart failure because neprilysin also degrades ANP/BNP.
• DPP-IV inhibitors (sitagliptin, saxagliptin) are used in type 2 diabetes because they prevent degradation of GLP-1.
• ACE inhibitors (captopril, enalapril) block angiotensin II formation and preserve bradykinin, used for hypertension and heart failure.

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4. Proteins and Neurotransmitters in Alzheimer's Disease

A. Amyloid Beta (Aβ) Plaques

• Precursor protein: Amyloid Precursor Protein (APP) - a transmembrane glycoprotein encoded by a gene on chromosome 21.
• Normal processing: APP is cleaved by α-secretase (non-amyloidogenic pathway) → produces soluble sAPPα.
• Pathological processing: APP is first cleaved by β-secretase (BACE1), then by γ-secretase (a complex containing presenilin-1 or presenilin-2) → produces Aβ40 and Aβ42 peptides. Aβ42 is more hydrophobic and prone to aggregation.
• Aβ peptides aggregate into oligomers (most neurotoxic form), then fibrils, then amyloid plaques (neuritic/senile plaques).
• Plaques accumulate extracellularly, also depositing in cerebral blood vessel walls (cerebral amyloid angiopathy).

B. Neurofibrillary Tangles (NFTs) - Tau Protein

• Tau is a microtubule-associated protein that normally stabilizes microtubules in neurons.
• In AD, tau becomes hyperphosphorylated due to an imbalance between protein kinases (GSK-3β, CDK5) and phosphatases (PP2A).
• Hyperphosphorylated tau detaches from microtubules → microtubules disassemble → free tau aggregates into paired helical filaments (PHFs) → NFTs form intracellularly.
• This disrupts axonal transport, causes neuroinflammation, and contributes to neuronal death.

C. Neurotransmitter Deficits

D. Other Key Proteins

• ApoE4 (apolipoprotein E4) - the major genetic risk factor for sporadic AD. ApoE4 impairs Aβ clearance and promotes tau phosphorylation. ApoE4 homozygosity increases risk ~12x vs. ApoE3.
• Presenilin-1 (PSEN1) and Presenilin-2 (PSEN2) - mutations cause familial early-onset AD by altering γ-secretase activity to overproduce Aβ42.
• Alpha-synuclein - primarily associated with Parkinson's but can co-occur in AD (Lewy body variant).

E. Current Drug Targets

• AChE inhibitors (donepezil, galantamine, rivastigmine): increase ACh by blocking acetylcholinesterase
• NMDA antagonist (memantine): blocks overactivated NMDA receptors to reduce glutamate excitotoxicity
• Anti-amyloid monoclonal antibodies (lecanemab, donanemab): remove Aβ plaques; slow but do not halt progression

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5. Catecholamine Transporters

5.1 Plasma Membrane Transporters (SLC6 Gene Family) - for Reuptake

5.2 Vesicular Transporters (SLC18 Gene Family)

5.3 Drugs That Increase (Block) These Transporters

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6. Function and Characteristics of Excitatory Neurotransmitters

Major Excitatory Neurotransmitters

A. Glutamate (Primary Excitatory NT of the CNS)

• Synthesis: From α-ketoglutarate (TCA cycle intermediate) via transamination by aspartate aminotransferase (AAT), or from glutamine by glutaminase. Glutamate synthesis directly "drains" α-KG from the TCA cycle and must be replenished via anaplerotic reactions (pyruvate carboxylase, methylmalonyl-CoA mutase).
• Storage: Vesicular glutamate transporters (vGluT1-3, SLC17 family) package glutamate into synaptic vesicles.
• Receptors:

• Inactivation: Reuptake via EAAT (Excitatory Amino Acid Transporters, SLC1 family) into presynaptic neurons AND astrocytes. In astrocytes, glutamate is converted to glutamine by glutamine synthetase → glutamine exported back to neurons (glutamate-glutamine cycle).
• Excitotoxicity: Excess synaptic glutamate (from energy failure, ischemia, hypoglycemia) causes prolonged NMDA receptor activation → massive Ca2+ influx → activation of destructive enzymes (calpains, endonucleases, nitric oxide synthase) → neuronal death. Key in stroke, TBI, hypoglycemia, and neurodegenerative diseases.

B. Aspartate

• Excitatory NT, but functions in fewer pathways than glutamate.
• Synthesized from the TCA cycle intermediate oxaloacetate via transamination.
• Acts on NMDA receptors (co-agonist with glutamate).
• Cannot cross the blood-brain barrier.

C. Acetylcholine (Excitatory at NMJ and ANS)

General Characteristics of Excitatory Neurotransmitters

1. Depolarize the postsynaptic membrane - move resting membrane potential toward the action potential threshold (more positive = more likely to fire)
2. Act on ionotropic receptors (fast, direct ion channel gating) or Gq-coupled metabotropic receptors (slow, via second messengers)
3. Increase intracellular Ca2+ (especially via NMDA) - critical for synaptic plasticity and gene expression
4. Terminated by reuptake (via EAATs for glutamate/aspartate) or hydrolysis (ACh via AChE)
5. Dual role: physiologic excitation is essential for all brain function; pathological excess causes excitotoxic neuronal death

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7. Function and Properties of Endocannabinoids

What Are Endocannabinoids?

• Synthesized on demand (not stored in vesicles) from membrane phospholipid precursors
• Released from the postsynaptic neuron (not presynaptic)
• Act in a retrograde direction - traveling backward across the synapse to inhibit the presynaptic terminal

Major Endocannabinoids

Receptors

Mechanism of Retrograde Signaling

1. Postsynaptic neuron is strongly depolarized (e.g., by glutamate) → Ca2+ enters → activates DAG lipase → 2-AG is synthesized from membrane DAG
2. 2-AG (or AEA) is released backward across the synapse
3. Binds to CB1 receptors on the presynaptic terminal
4. Gi activation → ↓cAMP and inhibition of voltage-gated Ca2+ channels → reduced Ca2+-dependent neurotransmitter vesicle release
5. Net effect: depolarization-induced suppression of inhibition (DSI) or excitation (DSE) → negative feedback control of synaptic strength

Key Functions

Properties Summary

• Lipid messengers (not water-soluble like peptides or amino acids)
• Synthesized on demand from arachidonic acid-containing phospholipids
• Retrograde messengers - unique among neurotransmitters
• Short half-life due to rapid degradation by FAAH (for AEA) and MAGL (for 2-AG)
• Pharmacological targets: THC (cannabis) is a partial CB1/CB2 agonist; FAAH inhibitors (under development) increase anandamide levels for pain/anxiety

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8. Mechanism and Symptoms of Parkinson's Disease

Core Pathological Mechanism

Biochemical Mechanisms

A. Dopamine Pathway Disruption

• The nigrostriatal dopamine pathway (SNpc → striatum) normally facilitates motor control by modulating the direct and indirect pathways of the basal ganglia.
• Loss of >60-80% of SNpc neurons and >80% of striatal dopamine is needed for symptoms to appear.
• Loss of dopamine disrupts the balance between the direct pathway (Go signal - normally facilitated by D1 receptors) and indirect pathway (Stop signal - normally inhibited by D2 receptors) → net overactivity of the subthalamic nucleus and GPi/SNpr → excessive inhibition of thalamus → reduced motor cortex activation

B. Alpha-Synuclein and Lewy Bodies

• The hallmark pathological finding is Lewy bodies - intraneuronal inclusions composed primarily of alpha-synuclein (misfolded and aggregated).
• Normally, alpha-synuclein is involved in vesicle cycling and dopamine release. When misfolded, it forms toxic oligomers → disrupts mitochondrial function, impairs proteasomal and lysosomal degradation → neuronal death.
• Mutations in SNCA (alpha-synuclein gene), LRRK2, Parkin, PINK1, and DJ-1 genes cause familial PD.

C. Mitochondrial Dysfunction & Oxidative Stress

• Dopaminergic neurons are especially vulnerable because dopamine metabolism via MAO generates H2O2 (hydrogen peroxide) → oxidative stress.
• Mitochondrial complex I deficiency is found in SNpc of PD patients → reduced ATP + increased reactive oxygen species → neuronal death.
• The neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a classic experimental model of PD: MPTP is converted to MPP+ by MAO-B → enters dopaminergic neurons via DAT → inhibits complex I → oxidative damage → selective SNpc neuron death.

D. Neuroinflammation

• Activated microglia surround Lewy body-containing neurons → release cytokines and reactive oxygen species → amplify neurodegeneration.

Symptoms

• Anosmia (loss of smell) - often the first symptom, due to early pathology in olfactory bulb
• REM sleep behavior disorder - acting out dreams (early sign)
• Constipation - enteric nervous system involvement
• Depression and anxiety - dopamine and serotonin deficits
• Cognitive decline/dementia (in advanced PD) - cholinergic neuron loss
• Autonomic dysfunction - orthostatic hypotension, hypersalivation

Treatment Targeting the Biochemical Defect

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Quick Reference Summary Table