ENZYME CHARACTERISTICS

• Increase rate of reaction by lowering activation energy
• Most are proteins
• Specific – conversion of one specific substance to one product
• May require cofactors or coenzymes
• Carefully regulated

ΔG = free energy

• Free energy of the product minus the free energy of the reactants
• ΔG is negative for enzymatic reaction because energy is released (exergonic reaction)

ΔEa = activation energy

• Energy barrier that must be overcome for a reaction to proceed
• Enzymes lower activation energy of a reaction by stabilizing transition state
• Energy required to get to equilibrium (rate of forward and reverse reactions are the same) correlates with ΔG and is unchanged in the presence or absence of enzyme
• Enzyme doesn’t dictate whether reaction will proceed but determines speed of reaction

ENZYME ACTIVE SITE

• 3D structure produces active site
• Shaped so that substrate fits in
• Product of an enzymatic reaction has lower affinity for binding site: exits binding site and is released

COFACTORS AND COENZYMES

• Bind cofactor binding site (distinct from active site)
• Some enzymes inactive without cofactor or coenzyme
• Many are vitamin-derived, metal ions, or other smaller organic molecules

PHENYLKETONURIA

Pathophysiology: Toxic Metabolites of Phenylalanine

To understand the pathophysiology of phenylketonuria, show that when phenylalanine accumulates at toxic levels, it transaminates into:

• Phenylpyruvate (aka phenyl ketone); hence, “phenylketonuria” describes the presence of phenylpyruvate, phenylalanine, and two key other derivatives in the urine and blood:
  • Phenylacetate which has a distinct “must/mousy odor”.
  • Phenyllactate.

Phenylalanine Excess / Tyrosine Deficiency

• Thus, overall indicate that in phenylketonuria, there is an:
  • Excess of phenylalanine
  • Deficiency of tyrosine

So the goal of therapy is to reduce phenylalanine intake and to supplement tyrosine deficiency via the diet. Remember the sparing action of tyrosine on the requirements of phenylalanine

Clinical Presentation of PKU

• Hypopigmentation
  • Indicate that hypopigmentation (of the skin and iris) is a finding in this disorder (remember: melanin is a derivative of tyrosine and tyrosine is deficient in PKU).
• Neuropsychiatric disorder
  • And because the toxic levels of phenylalanine and its derivatives are neurotoxic, this disorder causes tremo r, psychosis, seizures, and cognitive dysfunction.

PHEOCHROMOCYTOMA

Clinical Presentation of Pheochromocytoma

• Symptoms
  • Spontaneous severe anxiety: palpitations, sweating, panic
• Physical Exam Signs
  • Tachycardia (Rapid heart rate)
  • Hypertension (High blood pressure)

Biochemical Pathophysiology

As a simplification…

• Dopamine
  • We can attribute the agitation and possible psychosis to the surge in Dopamine.
• Norephine & Epinephrine
  • The sympathetic nervous system “fight or flight” symptoms relate to the surge in norepinephrine and epinephrine.

Laboratory Testing

• We test for pheochromocytoma in patients with unexplained episodic hypertension (high blood pressure) with blood and urine collection of:
  • Catecholamine metabolite levels
  • Metanephrine levels

• Specifically, we typically order:
  • Urine and plasma free metanephrines
  • Urine and plasma free catecholamines
  • Urine homovanillic acid (HVA)
  • Urine vanillylmandelic acid (VMA)

• The tests are highly sensitive, which leads to false positives. As anticipated, causes of false positives include:
  • Sympathetic nervous system agitation (ie, psychophysiological stress)
  • Exogenous triggers of catecholamines: pharmaceuticals, tobacco, caffeine, and illicit drugs.

Tumor Appearance

• Pheochromocytomas are black staining tumors (remember this was the color of melanin, another tyrosine derivative, as well) that classically grow out of the medullary layer of the adrenal gland.
  • For a better understanding of the difference between the adrenal medulla and the adrenal cortex, see adrenal gland hormone production.
• Paragangliomas are essentially extra-adrenal pheochromocytomas
  • They derive from cancerous autonomic nervous system tissue.
  • True to the anatomy of the autonomic nervous system – head/neck ANS paragangliomas are parasympathetic whereas thorax and abdominal paragangliomas are sympathetic.

PHARMACOTHERAPEUTICS IN PARKINSON’S DISEASE

Parkinson’s disease (and for that matter, all Parksinonism syndromes) are Dopamine deficiency syndromes within the brain.

Carbidopa

• Indicate that the pharmaceutical carbidopa is used to block the decarboxylation of DOPA to dopamine, peripherally, and increase the bioavailability of dopamine centrally (in the central nervous system – where it is intended to treat Parkinson’s disease).
• Dopamine cannot cross the blood brain barrier but DOPA can, so Dopamine is administered systemically as L-DOPA. However, if it were administered without a decarboxylase inhibitor (such as carbidopa) it would be decarboxylated peripherally into Dopamine and patients would simply become nauseated.
• In the presence of a peripheral decarboxylase inhibitor, DOPA is taken up in the CNS and THEN decarboxylated to Dopamine (in the basal ganglia where it serves to replenish the deficient stores of Dopamine). Carbidopa (itself) doesn’t cross the blood brain barrier.

Therapeutics

• Levodopa: Dopamine Precursor
  • So to treat a patient with Parkinson’s disease, let’s add levodopa as a Dopamine precursor.
• Carbidopa: DOPA decarboxylation Inhibitor
  • At the same time, we need to add Carbidopa to block the peripheral DOPA decarboxylation of DOPA – we need to ensure that the levodopa makes it through the systemic circulation and enters the brain, otherwise it will simply act like any catecholamine within the periphery and increase blood pressure and heart rate but fail to impact the central nervous system Dopamine deficiency state.
• Ropinirole & Pramipexole: Dopamine agonists
  • We can also add ropinirole or pramipexole, which are Dopamine agonists that optimize the release of Dopamine from the remaining Dopaminergic neurons within the substantia nigra.
• Entacapone: COMT Inhibitor
  • We can add entacapone, which is a COMT inhibitor to increase the circulation of the Dopamine that we’ve stimulated or replaced (ie, we can inhibit catecholamine metabolism).
• Selegeline or Rasagaline: MAO-B Inhibitors
• And we can add selegeline or rasagaline, which are MAO-B (specifically) inihibitors which also increase Dopamine but via MAO inhibition (catecholamine metabolism inhibition).
• The B subunit is specific to Dopamine catalysis, whereas the MAO-A enzyme is less specific and also metabolizes norepinephrine and serotonin, thus drugs that inhibit MAO-A are potentially much more hazardous to use.

HYPERTHYROIDISM

Clinical Presentation

• Hypermetabolic state that manifests with:
  • Weight loss, sweats, fevers, rapid heart rate.
• Skin and hair thinning
• Grave’s Ophthalmopathy
  • Ocular protrusion and reddening

ALKAPTONURIA

• Show alkaptonuria, which we can think of as a melanin-like substance in the urine and joints.

Pathogenesis

• It occurs from a deficiency in homogentisate 1,2 dioxygenase.
  • This results in a build-up of a melanin-like polymer called benzoquinone acetic acid (a product of the oxidation of homogentisic acid), which binds connective tissue and causes dark pigmentation or ochronosis (arthritis).
• Thus, we can think of alkaptonuria as the opposite of albinism + the build-up of acetic acid in the tissues irritates the joints and causes joint pain.

Presenting Symptoms

• Children: Dark Urine
  • The presenting manifestation in children is typically urine that darkens when it sits for awhile (not common now with disposable diapers). The darkening occurs from the excess homogentisate in the urine (5,000 mg vs 20-30mg (normally).
• Adults: Join Pain
  • In adults, the disease presents, typically, from joint pain from the build-up of acetic acid in the tissues, which irritates the joints.

TYROSINEMIA TYPE I

Pathogenesis: fumarylacetoacetate hydrolase deficiency

• Indicate that tyrosinemia type I (aka hereditary tyrosinemia, tyrosinosis) results from fumarylacetoacetate hydrolase deficiency.

Presenting symptom: “cabbage-like odor”

• Indicate that it characteristically causes a “cabbage-like odor” but importantly causes liver and kidney failure, polyneuropathy, and bone dysplasia (rickets), manifesting early-on with diarrhea, vomiting and tyrosine and its metabolites in the urine.
• Also consider that transient tyrosinemia (elevated blood levels of tyrosine) occurs in ~ 10% of newborns, most often due to vitamin C deficiency or immature liver enzymes due to premature birth.

STEPS OF VESICULAR BUDDING AND FUSION (+ PROTEINS INVOLVED)

• Cargo selection (cargo receptor, adaptor protein)
• Vesicular budding (adaptor proteins, coat proteins)
• Fission from donor membrane (dynamin)
• Vesicular coat dissociates
• Vesicular targeting and transport (Rab-GTPase, tethering protein)
• Fusion with target membrane (V-snare and T-snare)

PROTEINS OF VESICULAR BUDDING AND FUSION

• Cargo receptors – select and concentrate molecules to be transported in vesicle
• Adaptor proteins – bind cargo receptor and coat proteins
• Coat proteins – form protein scaffold around vesicle that facilitate facilitate vesicular budding
• Dynamin – GTPase involved in vesicular fission
• RabGTPase – associates with vesicle after coat has dissociated. Facilitates transport of vesicle to appropriate target membrane. Locks vesicle to target membrane by attaching tethering proteins
• Tethering proteins – anchored in target membrane, attach rabGTPase. Move vesicle close to target membrane for vesicular fusion.
• V-snares(vesicular) and T-snares(target membrane) – Play role in vesicular fusion

COAT PROTEINS DIRECT VESICLE TRANSPORT

• Clathrin +adaptin 1: Golgi → Lysosome
• Clathrin + adaptin 2: Plasma membrane → Endosomes (endocytosis)
• COP 1: Cis golgi → ER AND Later cisternae → Earlier ones (retrograde transport)
• COP II: ER → Cis golgi

3 PATHWAYS OF VESICULAR TRANSPORT

• Secretory pathway: delivers cargo to the plasma membrane.
• Endocytic pathway: uptake cargo from the plasma membrane.
• Retrieval pathway: recycles cellular molecules.

KEY FACTS ABOUT VESICULAR TRANSPORT:

• Compartment lumens mix via the transport intermediate.
• The membrane of each vesicle maintains its orientation.
• If the cell is growing, the secretory pathway is more active than the endocytic pathway.

STEPS IN SECRETORY PATHWAY

• Transport vesicles bud from the ER and carry content away from it to cis side of Golgi.
• Vesicular budding and fusion mediates the transport of cargo through the Golgi stacks, from cis to trans side.
• Cargo exits the Golgi via a transport vesicle on trans side.
• Transport vesicles fuse with plasma membrane or with endosomes (and then lysosomes).

STEPS IN ENDOCYTOTIC PATHWAY

• Early endosome forms from plasma membrane and extracellular materials.
• Early endosome targets cargo to late endosomes.
• Late endosomes then deliver cargo to lysosomes, which degrade cargo.

THE RETRIEVAL PATHWAY TAKES SEVERAL FORMS

• Endosomes can return cargo to the cell surface via recycling endosomes.
• Cargo in early and late endosomes can also return to the Golgi for reuse.
• Vesicles can deliver proteins from the trans face to the cis face of the Golgi.
• Vesicles can return proteins from the golgi to the ER as well.

3 STEPS OF VESICULAR FORMATION

• Cargo selection. Incorporation of cargo into a vesicle is carefully regulated to ensure that only the correct cargo gets transported.
• Vesicular budding. deformation of the hydrophobic membrane bilayer and breaking off of the membrane into a vesicle
• Vesicular targeting and fusion. Highly regulated just like cargo selection.

Cellular compartments are topologically equivalent when:

• Molecules can get from one to another without having to cross a membrane.
• Nuclear envelope, ER, Golgi, transport vesicles, endosomes, lysosomes, and extracellular space = topologically equivalent