Nitrogen Metabolism - L11 - 10/01

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What starts protein digestion and where?
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Terms in this set (44)
What starts protein digestion and where?
Low pH of the stomach denatures proteins
How is pepsinogen cleaved? What are pepsin's functions?
Autocatalytically

Pepsin protease cleaves peptide bond on denatured proteins

Functions as:

Endopeptidase - breaks peptide bonds on non-terminal amino acids

Exopeptidase - breaks peptide bonds on terminal amino acids
Where does protein digestion occur after the stomach?
Protein digestion continues in the small intestine

Exocrine pancreas secretes enzymes including brush border enzymes (aminopeptidases) into small intestine to degrade proteins

Bicarbonate secreted to raise pH
Describe proteases responsible for digestion, their general substrate preference, and their enzymatic activation.
Aminopeptidase: cleaves at N-terminus

Proenzymes (zymogens) → Active enzymes

Pepsinogen -H+→ Pepsin
- Phe, Tyr, Glu, Asp
- Aromatic + (+)

Trypsinogen -enteropeptidase→ Trypsin
- (+) charged
- Arg, Lys (Basic)

Chymotrypsinogen -Trypsin→ Chymotrypsin
- Bulky, Aromatic
- Phe, Tyr, Trp, Leu

Proelastase -Trypsin→ Elastase
- Small, non-charged
- Ala, Gly, Ser

Procarboxypeptidase -Trypsin→ carboxypeptidase
- Carboxypeptidase A (hydrophobic AAs)
- Carboxypeptidase B (Arg, Lys)
- Cleaves C-Terminus
Image: Describe proteases responsible for digestion, their general substrate preference, and their enzymatic activation.
Describe how protein digestion is affected in cystic fibrosis and its clinical presentation. Treatment?
Thickening of exocrine secretions can cause blockage which can affect secretion of digestive enzymes into the intestinal lumen

- Stunted growth, foul-smelling stools

- Treated w/ pancreatic enzymes (inc. conc. w/ age)

- Affects secretion of lipase & pancreatic amylase; limits digestion of Carbs & fats
Explain how amino acids are absorbed by the body
From Gut to epithelial cell:
Cotransport of Na+ & amino acids

From epithelial cell to blood:
Facilitated amino acid transport
Image: Explain how amino acids are absorbed by the body
1. Describe the biochemical basis of Hartnup Disease

2. How is it detected?

3. What symptoms may arise as a result of what specific amino acid?

4. Treatment
1. Defect in B⁰ transport of neutral amino acids

2. ↑ a.a. in urine (may be the only sign)

3. May develop pellagra-like symptoms if Trp deficient. Trp is NAD⁺ precursor

4. Niacin & high-protein diet
1. Describe the biochemical basis of Cystinuria

2. Treatment
1. Defect in B⁰⁺ transport of basic amino acids (Cys, Arg). Decreased resorption of aa from glomerular filtrate results in an ↑ cys insolubility > renal stones

2. ↑ Fluid intake & ↑ urine pH via medication
1. What contributes to the amino acid pool?

2. What enzymes are involved with intracellular protein digestion?

3. Describe Ubiquitin-Proteasome System
1. Dietary and intracellular proteins

2. Lysosomes & Ubiquitin-Proteasome System

3. Ubiquitin-Proteasome System
20S proteasome binds w/ 19S regulatory particle (can bind w/ two of em) forming 26S proteasome; Requires ATP for proteolysis
Image: 1. What contributes to the amino acid pool? 2. What enzymes are involved with intracellular protein digestion? 3. Describe Ubiquitin-Proteasome System
Kwashiorkor
Protein deficiency that may have adequate caloric intake however a negative N balance due to lack of essential a.a.

Clinical Presentation
- Inability to synthesize necessary protein > edema, hypoalbuminemia, potbelly
- Anemia
MarasmusProlonged protein and energy malnutrition (inadequate caloric intake) Clinical Presentation - Slowed growth/ no muscle - Emaciated (skin and bones)Fate of amino acids in fed stateMost dietary a.a. used for protein synthesis, excess converted & stored as glycogen and TGs (VLDL)Fate of amino acids in fasting stateAmino acids released from proteolysis are used as a fuel source. - C used for fuel - N excreted as UreaWhat is the importance of glutamine and alanine in nitrogen transport?Glutamine and alanine are major N carriers in the blood (skeletal muscle exports) Alanine is the major gluconeogenic a.a.What is the fate of amino acid carbons and nitrogen?- C skeleton used as fuel - N excreted because NH₃ is toxicWhere does the N in Urea come fromNH₄⁺ & AspDescribe transamination reactions (including cofactor used) and defend their importanceTransfer amino group from amino acid to α-Keto Acid via PLP cofactor (Vit. B₆ Precursor) Important for the funneling of N to select amino acids.Explain the role of the glutamate dehydrogenase reaction in nitrogen metabolism, and identify the cofactor used.Releases NH₄⁺ from amino acid and reduces NADP to NADPHNitrogen Funneling SummaryGlutamate found/ produced in 3 different ways 1. Glu (Already an amino acid) 2. AA + α-kg > α-ka + Glu (PLP-TransAm) 3. α-kg + NADPH + H+ > Glu (Reverse GDH using NADPH) Glutamate funnel N in 4 different ways 1. Glu + GDH > α-kg + NH4+ > Urea Cycle (GDH) 2. OAA + Glu > Asp > Urea Cycle (PLP-TransAm) 3. Glu + Pyruvate > α-kg + Ala > C + (N > Urea Cycle) (PLP-TransAm) 4. Glu > Gln (Gln Synthetase) > liver > Gln (Glnase)> Glu > Glu (GDH)> α-kg + NH4+1. Describe the clinical consequences of hyperammonemia 2. Treatment1. Irreversible brain damage and mental retardation 2. Diagnose early, treat aggressively with N-removing compounds, low-protein diet.Urea cycle purpose? Identify its enzymes and intermediatesExcretes N as urea because NH3 is toxic to the brain 1. HCO3- + NH4+ -CPS1→ CP 2. CP + Orn -Orn-TransCMlase→ Citr (Mit) 3. Citr (Mit) > Citr (Csol) 4. Citr + Asp + ATP -ASStase→ ArgSuc 5. ArgSuc -ASLyase→ Fum + Arg 6. Arg -arginase→ Urea + Ornithine 7. Urea goes to urine 8. Ornithine goes back to cycle Synthetase rxns utilize 2 ATPHow is the urea cycle regulated?- Arg stimulates Glu + Acetyl CoA rxn to form NAG - NAG allosterically activates CPSI rxnWhat happens if there is a urea cycle enzyme defect or deficiency? What is the most common urea cycle defectBuild up of ammonium (toxic to the CNS) OTC deficiencyDescribe how orotate in urine is indicative of a urea problem1. OTC deficiency leads to accumulation of ammonia, Gln, and carbamoyl phosphate (CP) 2. CP is a substrate for pyrimidine synthesis 3. Pyrimidine synthesis pathway becomes flooded, excess orotate produced 4. Excreted in urine This excess orotate is indicative of a problem in the urea cycleTreating urea cycle disordersN-removing compounds Drugs form conjugates w/ AAs (facilitate excretion), uses up N so body replaces those that were excreted - Benzoic Acid - PhenylbutyrateWhat are essential amino acids?AAs required from dietary sources VHIMKRL (vehicle) FTW (for the win)What are nonessential amino acids?Synthesized in the body CANDY EQ (equilibrium) GPSGlucogenic & Ketogenic Amino acidsGlucogenic: C skeletons converted to some product of glucose breakdown Ketogenic: C skeletons converted to acetyl-CoA or acetoacetateCystinuriaDisorder caused by defective transport proteins - Cys is not adequately reabsorbed from glomerular filtrate - Insoluble Cys forms renal calculiHomocystinuria What molecules are high? Cause, Types, TreatmentMet and Hcy are high, Cys is low Cause: autosomal recessive defect in Cystathionine β-Synthase (more common) or deficient cystathionase (measure serum cystathionine to distinguish); could also be inadequate intake of dietary folate or B12 Most common - Myopia and dislocation of lens - Increased risk of abnormal blood clotting - Brittle bones or other skeletal abnormalities (long bones) - ~ Developmental delay & learning problems Less common - Intellectual disability - Problems w/ movement - Failure to thrive - Seizures - Megaloblastic anemia Treatment - Early reduction in blood Met & Hcy - Low Met Diet, high doses of pyridoxine (B6) - If any signs of vascular events, seek immediate helpMaple Syrup Urine Disease Cause & SymptomsCause: - branched-chain α-Ka DH is defective - BCAAs and α-keto acid accumulate and appear in urine - Symptoms - Sweet odor of urine - Poor Feeding, vomiting - Lethargy - Abnormal Movements - Delayed Development - Seizures, coma, and death possible w/o treatmentProblems w/ Phe and Tyr metabolism can cause what 3 clinical correlates- Phenylketonuria (PKU) - Alkaptonuria - TyrosinemiaPhenylketonuria (PKU) Cause, Symptoms, TreatmentCause: Autosomal recessive defect in phenylalanine hydroxylase (PAH) leads to hyperphenylalaninemia Symptoms - Irreversible mental retardation - Delayed psychomotor skills - Musty or mouse-like odor to urine (excess Phe) - Tend to have lighter skin and hair - Microcephaly - Likely to have skin disorders - Tremors & Seizures - Hyperactivity Treatment - Maintain Phe levels in blood through dietAlkaptonuria Cause & symptomsCause Homogentisate oxidase is defective causing accumulation of Homogentisate, which auto-oxidizes to a dark pigment that is excreted in urine Symptoms - Ochronosis: buildup of dark pigment in connective tissue such as cartilage and skin, as well as in sclera of eyes - Often develop arthritisTyrosinemia Types & symptomsCause Type I: Genetic defect in fumarylacetoacetate hydrolase - Fatal w/in first year of life - Multi-organ failure Type II: Defect in Tyr aminotransferase - Tyrosine accumulation - Intellectual disability - Eye Pain, redness, photophobia - Thick, painful skin on palms, solesWhich amino acid may serve as a precursor for NAD(P)⁺ synthesis? A deficiency in this amino acid may result in what? give an example of such a disorderTryptophan > Vit. B₆ (Niacin) > NAD(P)⁺ Inadequate Trp > Pellagra - Dermatitis, diarrhea, dementia, death Hartnup DiseaseWhat are the roles of purine and pyrimidines in the body- DNA and RNA - Cofactors (NAD+, FAD, CoA) - Energy (ATP/ GTP) - Activated intermediates in biosynthesis - SAM for methyl transfer - 2nd messengers (cAMP) - Allosteric regulators in metabolismWhat molecules contribute to purine synthesis?N¹⁰-formyl-FH₄ transfers single C groups (Derived from folate (Vit. B9)) Folate is needed for purine (and therefore DNA) synthesis Gln & Asp required for base modificationDescribe the importance of PRPP, IMP, ATP, GTP, Asp, and Gln in purine synthesis- PRPP (Phosphoribosyl Pyrophosphate) is activated ribose - IMP is branch point - GTP is required to make AMP - ATP is required to make GMPPurine Synthesis Pathway1. R5P + ATP > PRPP 2. PRPP + Gln >>> IMP 3a. IMP + GTP + Asp >> dATP/ ATP 3b. IMP + ATP + Gln >> dGTP/ GTP Regulation of purine synthesis enzymes by downstream productsPyrimidine SynthesisBase made first Aspartate + CO2 + Gln > Base then attached to R5P (CPSII)> Carbamoyl Phosphate > Orotate > pyrimidine CPS-II similar to CPSI rxn in urea cycle but: - Occurs in cytosol (not mitochondria) - Uses Gln instead of NH4+ - Regulated stepWhat does pyrimidine synthesis have to do with the urea cycle?Urea cycle defect in OTC results in accumulation of Carbamoyl phosphate, which means some ends up in cytosol, where it is available to enzymes in pyrimidine synthesis pathway, leading to increase orotate production (which ends up in urine). This is different from hereditary orotic aciduriaOTC Enzyme Deficiency vs Hereditary Orotic AciduriaOTC Enzyme Deficiency - Urea cycle disorder - Ornithine transcarbamoylase deficiency - Pyrimidines can still be made - NH4+ accumulates, toxic to CNS - Treat urea cycle defect w/ N-removing compounds, low protein diet Hereditary Orotic Aciduria - Orotate phosphoribosyltransferase & orotidine 5' phosphate decarboxylase are defective - Pyrimidines cannot be made - Normal growth doesn't occur - Treat pyrimidine synthesis defect w/ oral administration of uridineGout Cause & TreatmentCause Overproduction or decreased excretion of purine > increase Purine degradation > increase uric acid in blood > precipitation > tissues/ joints inflamed. Treatment - Allopurinol - xanthine oxidase inhibitor - Avoid diet rich in meat, seafood, fructose-sweetened drinks, alcohol