Biochem - Proteins, AA Catabolism vs Urea Cycle Flashcards
Conventionally a peptide chain is written in what direction?
The opposite as it is synthesized, starting with the N terminus
Synthesis starts with the C-terminus
Cathode and Anode in electrophoresis
Chathode (-)
Anode (+)
Alpha Helix
-NH H-bond every four AA’s along the chain
Beta Sheet
H-bond between C=O and -NH
Secondary Stx Motifs
Helix-turn-helix
Leucine zipper
Zinc finger
Found within transcription factors
Structural subunit of F-actin
Oligomer
Enthalpy of protein folding
Essentially isothermic (change in enthalpy is very small)
Although many H-bonds are broken during protein formation, the same number are broken from water
What energetically drives protein folding?
Entropy - resulting from increased disorder of water molecules as hydrophobic regions associate inside the protein
Molten globules
Intermediate between secondary structures and the folded protein
Heat shock proteins
Chaperones - prevent the aggregation of newly synthesized proteins by binding to their hydrophobic regions
I.e. HSP10, HSP 60, and HSP70
Myoglobin vs. Hemoglobin
Myoglobin - single peptide with one O2 binding site
HbA - tetrameric with two alpha and two beta polypeptide chains (four binding sites) each conjugated with a heme group.
R state
Hemoglobin
Relaxed state
When one O2 molecule binds to a heme of hemoglobin
Increases affinity of O2 binding
Positive cooperativity
What reduces the O2 affinity of hemoglobin
Acidity (increased conc. of CO2)
This is called the Bohr effect
Binding of 2,3-bisphosphoglycerate (BPG)
Decreases the affinity of HbA for O2
BPG is by-product of glycolysis, this ensures efficient O2 offloading in tissues that are metabolically active, especially in high altitude or anemia, where intracellular BPG levels might be elevated
Sickle-cell anemia
The beta chain of hemoglobin undergoes a point mutation that causes a missense (E6V) mutation in the reading frame
The introduction of hydrophobic valine promotes polymerization of deoxygenated mutant hemoglobin
HbS polymers adopt the classic “sickle” shape - prone to hemolysis leading to anemia
Peptide configuration of collagen
Gly - X - Y
Gly - every third amino acid
X - typically proline
Y - typically hydroxyproline or hydroxy lysine
Collagen is a triple helical structure, where three polypeptide chains intertwine around each other
Synthesis of collagen
pro-collagen syn. in rER, where the signal sequence (pre) is removed)
In the rER lumen, pro and lys residues undergo hydroxylation (depends on presence of oxygen and Vit C)
Hydroxylysine residues are further glycoslated with galactose and glucose
Modified peptide chains are assembled into the triple helix, forming procollagen, which is secreted outside the cell and processed to form mature collagen
Collagen fibers undergo cross-linking by oxidation of amino acid side chains
EDS
Ehlers-Danlos Syndrome
Mutations in collagen synthesis and processing - fragile skin and hyper-elastic
Joint hypermobility
Osteogenesis imperfect
Mutations in collagen I genes
Brittle bones which are prone to fracture
Scurvy
Impairs the hydroxylation of proline and lysine residues
Results in unstable collagen (mp from 42 C to 24 C) as the result of the loss of interstrand H-bond fomration from lack of pro-OH
sxs - bleeding gums and poor wound healing
Alport syndrome
Mutations in collagen IV disrupts the collagen network supporting kidney glomerular cells
Leads to compromised kidneys and failure
Proteopathies
Diseases classified by misfolded proteins
CJD
Creutzfeldt-Jakob Disease - average onset at 68 y/o
Progresses rapidly - death within year
Rare
vCJD
Variant Creutzfeldt-Jakob Disease - younger-onset form, avg. death age of 28 y/o
Linked to consumption of beef from cows affected by bovine spongiform encephalopathy
Kuru
Prion disease ID’d among the Fore tribe in Papua New Guinea
Believed to have spread through cannabilistic practices
PrPc
Normal prion proteins - are primarily alpha-helical structures in the brain
PrPSC
Misfolded, disease-causing conformation of the prion, pirmarily beta-sheet conformation
Propagation of prion disease
The misfolded PrPSC acts as a template, causing endogenous PrPC to also misfold
Prion disease transmission
After consumption, PrPSC crosses the intestinal lining, gets taken up by immune cells, and is transported to the brain
Affect of prions disease in the brain
Accumulation of beta-sheet rich prions leads to the formation of protein plaques in the brain
Hallmark of prion disease
Alzheimer’s
Buildup of misfoled Aβ42 protein in the brain
APP undergoes enzymatic cleavages - resulting in Aβ42 fragments that can aggregate and form amyloid or senile plaques outside neurons
Much like prions, Aβ42 plaques adopt a beta-sheet conformation, leading to the formation of insoluble aggregates (resists degradation through normal cellular processes)
Intracellular tangles of tau (Aβ42 causes intracellular phosphorylation of tau protein) forms neurofibrillary tangles - believed to damage neurons from within
Aβ42 originates from amyloid precursor protein (APP) - transmembrane protein believed to play role in neuron growth, survival, and repair post-injury
Genetic factors of Alzheimer’s disease
Mutations in the APP gene and componenets of the gamma-secretase enzyme (involved in APP cleavage) have been linked to increased Aβ42 production
A patient with a history of recurrent vaso-occlusive crises is found to have a mutation in the β-chain of hemoglobin. Which of the following amino acid substitutions is most likely responsible for this patient’s condition?
Glutamate to valine (E6V)
What sequence of AA residues is most commonly found in collagen?
Gly-X-Y
X - typically proline
Y - either pro-OH or Lys-OH
Difference between amyloid plaques and neurofirillary tangles?
Aβ42 plaques - outside of neurons
Neurofibrillary tangles - inside of neurons (Aβ42 phosphorylates tau causing it to tangle)
A deficiency in what vitamin impairs the hydroxylation of proline and lysine residues in collagen synthesis?
Vitamn C (Scurvy)
Active site
Michaelis-Mentin Eq
Km
Affinity of enzyme to substrate (lower Km = higher affinity)
Mathematically derived as the substrate concentration at which v = 1/2(vmax)
When [S] «< Km
How is v affected?
The rate is directly proportional to [S]
When [S]»_space;> Km
The rate approaches Vmax and becomes independent of [S]
Cofactors
Inorganic ions or organic molecules that bind to enzymes and are essential for activity
Types:
Posthetic groups and loosely bound cofactors
Fx - assist substrate binding, stabilize enzyme stx, or participate directly in reaction by facilitating transfer of chemical groups or electrons
Prosthetic Groups
Tightly and permanently bound to the enzyme (i.e. heme is a prosthetic group of hemoglobin)
Type of cofactor
Loosely Bound Cofactor
Can associate and dissociate from the enzyme (when bound, activate the enzyme)
Coenzymes
Subset of cofactors that are organic molecules - often derived from vitamins
Unlike cofactors, coenzymes are not permanently bound to the enzyme
Fx - carriers, transporting specific groups or electrons from one reaction to another (many redox rx’ns)
i.e.
NAD+ and NADP+ (derived from niacin)
CoA (derived from panthenic acid)
FAD (derived from riboflavin)
Affect of cofactors/coenzymes on enzyme
Catalytic activity
Specificity
Regulation (allosteric)
Diversity of rx’ns
Cooperativity
The binding of a substrate to one active site can affect the binding of substrates to other active sites
Allosteric enzymes often display _____ kinetics when rx’n velocity is plotted against [S]
Sigmoidal
Affects of allosteric regulation
- Fine-tuning of metabolic pathways
- Feedback inhibition
- Rapid response
- Integration of mlutiple signals
Competitive inhibitors
Can be overcome by adding more substrate (increasing [S])
Ex: Statins competitively inhibit HMG-CoA reductase
Vmax remains unchanged
Km increases (a higher [S] is need to achieve half of Vmax)
Noncompetitive inhibitors
Bind to allosteric site, causing conformational change
Increasing [S] does not overcome inhibition
Ex: heavy metals like lead or mercury
Vmax decreases
Km remains unchanged (affinity is not affected)
Lineweaver-Burke
Enzyme inhibition graphs
Graphs of V vs [S] for an allosteric enzyme
Does not follow MM kinetics, but is hyperbolic and the v vs. [S] is sigmoidal
Inborn errors of metabolism
PKU, galactosemia, and Tay-Sachs disease
Accumulation of Phe
PKU - deficiency of phenylalanine hydroxylas leads to accumulation of phenylalanine which causes intellectual disability if not managed
G6PD deficiency
glucose-6-phosphate dehydrogenase deficiency can lead to hemolytic anemia (especially after exposure to certain drugs or infections)
Disorder of purine and pyrimidine metabolism
Can lead to conditions like Lesch-Hyhan syndrome or orotic aciduria
Congenital adrenal hyperplasia
An endocrine disorder that can result from deficiencies in enzymes involved in cortisol synthesis
Where are AA’s abosrbed
Intestinal epithelial cells and taken up into cellular AA pools
Essential AA’s
Cannot be made and must be obtained through diet
PVT TIM HaLL (Phe, Val, Trp, Thr, Ile, Met, His, Lys, Leu)
a = arginine (conditionally essential during periods of rapid growth)
Primary reserve of AA’s
Muscle (this includes reserve for essential AA’s)
Diets deficient in essential AA’s can cause muscle wasting (when diet is deficient in essential AA’s, it can take up to six weeks for sxs to manifest due to muscle reserves)
Kwashiorkor malnutrition
Protein deficiency with adequate calories
Children develop swollen belly (ascitic belly) becuase albumin is 97% of serum protein and is responsible for oncotic pressure
Fluid collects in abdomen due to gravity
Marasmus malnutrition
Inadequate energy intake in all forms, including protein
Cystinuria
Autosomal recessive
Impairs the reabsorption of dibasic AA (disulfide linked Cys-Cys, ornithine, arginine, and lysine)
Hexagonal crystals of cystine in urinalyses are diagnostic of cystinuria
Recurrent nephrolithiasis (kidney stones)
Management:
Low cystine diet and increased fluid intake
Potassium citrate and sodium bicarb (make urine less acidic and increases solubility of cystine)
Pencillamine and topronin are cystine-binding thiol drugs that bind and form a water-soluble complex
Hartnup Disease
Autosomal recessive
Affects renal tubule reabsorption of neutral AA’s, importantly tryptophan
Trp is used to synthesize B3 (niacin) so sxs resemble deficiency of niacin (pellagra)
Management:
Dietary intake of trp-rich foods, trp and niacin supplements
3 D’s of Pellagra
Dermatitis
Diarrhea
Dementia
(Death)
Other malabsorption disorders and chronic alcoholism can present with pellagra like sxs
Corn-based diets (low in trp and niacin) increase risk of pellagra
Hartnup vs. Cystinuria
Ammonia toxicity
Toxic, especially to the CNS (causing encephalopathy)
When ammonia levels are high, astroctyes convert excess into glutamine, which is osmotically active. This draws water into astrocytes leading to cellular swelling and cerebral edema
Disposal of ammonia
80-90% of ammonia is disposed via formation of urea in the liver and excreted by the kidneys
Carried in blood by alanine (muscle) and glutamine (most tissues)
What are the most abundant AA’s in the blood?
Alanine (muscle) and glutamine (most tissues)
Transport ammonia to the liver
Enzymes of AA catabolism
Amino transferases (aka transaminases) - transfer NH2 from an AA to another alpha-keto acid, which is converted to its cognate AA
Requires pyridoxal phosphate (PLP) a B6 derivative, as a cofactor
Reversible to allow interconversion of AA and ketoacids as needed for metabolic need
Specific aminotransferases for each AA (except thr. and lys)
-Most use alpha-ketoglutarate and glutamate
Thus amino groups are funneled i nto glutamate by transamination reactions
ALT
Crucial in the glucose-alanine cycle (allows the body to transport nitrogen and carbon between muscles and the liver to maintain blood glucose levels during fasting or exercise, providing a way to remove excess nitrogen from muscle tissue.
In muscle, when AA are broken down their amino groups are transferred by aminotransferases to alpha-KG to generate Glu. ALT then transfers the amino from Glu to pyruvate producing Ala.
Ala is released into the blood and travels to the livers
The liver converts Ala back to pyruvate and Glu
The pyruvate can be used to produce glucose
The amino group from glutamate is used to form urea
AST
Aspartate transaminase
Where are ALT and AST highly expressed?
Liver
Also expressed in heart, muscle, and kidneys
Glutamine Synthetase
Glu + NH3 +ATP -> Gln + ADP + Pi
GS fixes free NH3 to glutamate (produced by transaminases) to make glutamine which is released into the bloodstream for transport to liver
Kidneys and brain also take up Gln for metabolic use
Glutaminase
Releases NH3 from glutamine to regenerate glutamate
In the liver, the released NH3 enters the urea cycle
Glutamate is deaminated again by glutamate DH which regenerates alpha-ketoglutarate
Glutaminase in kidney
Releases NH3 from glutamine to help regulate urine pH
Glutaminase function in the brain
The formation of glutamate from glutamine via glutaminase is a pathway used to form the excitatory neurotransmitter (glutamate)
Glutamate DH
Deaminates glutamate to form alpha-ketoglutarate/NAD(P)H/NH3
Reversible rx’n that is used to fix free ammonia to form glutamate
Why is glutamate unique?
Only AA that undergoes rapid oxidative deamination
In the liver, what enzymes release ammonia to enter the urea cycle?
Glutaminase and glutamate DH
Carbomyl Phosphate Synthetase I
Step 1 of urea cycle
Rate-limiting step
N-acetyl glutamate is essential allosteric activator of CPS-I (formed by NAG synthetase which is activated by R)
Mitochondrial enzyme
Why does adding arginine dietary intake help Tx Pt’s with some urea cycle disorders?
Arginine activates N-acetyl glutamate synthetase, which allosterically activates CPS-I
Ornithine Transcarbamoylase (OTC)
Step two of urea cycle
carbamoyl phosphate + ornithine -> citrulline
Mitochondrial enzyme
Argininosuccinate Synthetase (AS)
Step three or urea cycle
citrulline + aspartate -> argininosuccinate
AST supplies aspartate for this step
Argininosuccinate lyase
Step four of urea cycle
argininosuccinate -> arginine + fumarate
Arginase
Step five of urea cycle
arginine -> urea + ornithine
Hyperammonemia
Causes:
Liver dysfunction
Urea cycle disorders (UCD)
Sxs: asterixis (flapping tremor), vomiting, confusion, blurred vision, slurred speech, seizures
Only effective means to rapidly reduce level of circulating ammonia
Hemodialysis
Ammonia scavengers
Form water soluble products with gly or glu that are excreted by the kidneys, providing alternate pathway for removal of NH3
Sodium benzoate - combines with gly in liver to form hippuric acid which is excreted
Sodium phenylbutyrate - metabolized to phenylacetate which reacts with gln to form phenylacetylglutamine which is excreted
Lactulose
Non-absorbable disaccharide used in context of liver diseases. Fermentation of lactulose by gut bacteria lowers colonic pH.
Lower colonic pH promotes conversion of ammonia to ammonium, which do not easily diffuse across gut epithelium
NH3 is then able to be excreted in feces
Rifaximin
Abx used to reduce the population of ammonia-producing bacteria in the gut
Long-term management of hyperammonemia
Low-protein diet
Ammonia scavenger meds
Liver transplantation
Urea cycle disorders
A complete lack of any enzyme of the urea cycle results in death shortly after birth
Deficiencies present after first feeding (first protein rich meal)
Deficiency of urea cycle enzyme:
Increased serum levels of gln
CPS-I deficieCPSD)
Aka hyperammonemia-I
Most severe UCD
A 30-year-old woman is concerned about her pregnancy after learning that her brother has a urea cycle disorder. She is currently 10 weeks pregnant and asymptomatic. Which of the following tests would be most appropriate to screen for the carrier status of ornithine transcarbamylase deficiency in this patient?
- Serum ammonia level
- Urinary orotic acid after protein load
- Plasma citrulline level
- Liver biopsy
- Serum urea nitrogen level
Urinary orotic acid after protein load.
Rationale: This test is most appropriate for screening for carrier status of ornithine transcarbamylase (OTC) deficiency. OTC deficiency is an X-linked disorder, and female carriers can be asymptomatic. The protein load test stimulates the urea cycle, and in OTC deficiency carriers, this leads to increased orotic acid excretion due to the accumulation of carbamoyl phosphate, which is then shunted into the pyrimidine synthesis pathway.
A 28-year-old man presents to the clinic with a history of recurrent skin rashes, particularly on sun-exposed areas. He also complains of intermittent diarrhea and occasional confusion. Physical examination reveals a rough, scaly rash on face and hands. Which of the following amino acids is most likely affected in this condition?
- Phenylalanine
- Tryptophan
- Leucine
- Methionine
- Arginine
Tryptophan
Rationale: The clinical presentation of recurrent skin rashes on sun-exposed areas, intermittent diarrhea, and occasional confusion is consistent with Hartnup disease. Hartnup disease is a disorder of neutral amino acid transport, particularly affecting tryptophan absorption. Tryptophan is a precursor for niacin (vitamin B3), and its deficiency leads to pellagra-like symptoms, which include dermatitis, diarrhea, and dementia (the “3 D’s” of pellagra).
A newborn presents with lethargy, poor feeding, and respiratory distress within 48 hours of birth. Laboratory tests reveal elevated plasma ammonia and trace amounts of citrulline. Urinary orotic acid levels are normal. Which of the following is the most likely diagnosis?
- Ornithine transcarbamylase deficiency
- Argininosuccinate synthetase deficiency
- Carbomoyl phosphate synthetase I deficiency
- Argininosuccinate lyase deficiency
- N-acetylglutamate synthase deficiency
Carbamoyl phosphate synthetase I deficiency
Rationale: The presentation of hyperammonemia within 48 hours of birth, along with trace amounts of citrulline and normal urinary orotic acid levels, is consistent with carbamoyl phosphate synthetase I (CPS-I) deficiency. CPS-I catalyzes the first step of the urea cycle. Its deficiency leads to ammonia accumulation and very low or absent citrulline. Normal orotic acid levels distinguish it from ornithine transcarbamylase deficiency, which would show elevated orotic acid.
A 45-year-old man with a history of chronic alcoholism presents with confusion, asterixis, and a “sweet” odor on his breath. His blood ammonia level is 180 μmol/L. Which of the following medications would be most appropriate for immediate management?
- Sodium benzoate
- Rifaximin
- Lactulose
- Carbaglue (carglumic acid)
- Penicillamine
** Lactulose**
Rationale: The patient presents with signs of hepatic encephalopathy secondary to chronic alcoholism. Lactulose is the first-line treatment for acute management of hepatic encephalopathy. It works by lowering colonic pH, which promotes the conversion of NH3 to NH4+ ions, reducing ammonia absorption from the gut. The “sweet” odor on the breath is likely fetor hepaticus, often associated with severe liver disease.
A patient with recurrent kidney stones is diagnosed with cystinuria. Which of the following is the primary defect in this condition?
Impaired reabsorption of dibasic amino acids in the kidneys
Rationale: Cystinuria is characterized by impaired reabsorption of dibasic amino acids (cystine, ornithine, arginine, and lysine) in the kidneys. This leads to excessive excretion of these amino acids, particularly cystine, which has low solubility and tends to form kidney stones. The primary defect is in the transport system for these amino acids in the renal tubules, not in their metabolism or production.
Drug company develops a therapy to treat a cell membrane protein misfolding disorder. This mutation leads to abnormal protein folding and subsequent intracellular degradation of the protein before it can reach the cell membrane. With the new combination of drug therapy, the first drug corrects the processing and trafficking of the protein, enabling it to reach the cell surface membrane. Once the protein has reached the cell surface, its function is enhanced by the second drug. This therapy is most likely to be helpful in which of the following conditions?
Cystic fibrosis
Atrophy of CNS is common in many pathologies. What type of CNS atrophy is unique to Creutzfeldt-Jakob disease?
Atrophy of the cerebellum
Steps of the urea cycle
Remember enzymes by: COAAA
Specific sxs of osteogenesis imperfecta
Transulcent skin showing visible underlying veins
Frequent bone fractures
Col I deficiency
Specfic sxs of scurvy
Bleeding from gums
Activator of NAG
Arginine
NAG is an obligate activator of CPS I
OTC deficiency inheritance pattern
X-linked recessive
Only x-linked disease in the urea cycle
Female carriers report aversion to high-protein foods (due to lionization of x-chromosome)
