Nitrogen Flashcards
What are the different N-containig compounds?
- Metabolites
- Purines
- Heme = Succinyl-CoA + Glycine
- Hormones
What is the role of Nitrogenase?
Expressed by Diazotroph bacteria → fixes atmospheric nitrogen
Turns stable N2 gas into NH3
N2 + 8H+ + 8e- + 16 ATP + 16 H2O → 2 NH3 + H2 + 16 ADP + 16 Pi
*requires ATP to reduce these stable bonds (triple bond)
*FIXATION
What is the Haber-Bosch reaction?
N2 + 3H2 → 2 NH3
*In bacteria in the ground
What is assimilation of Nitrogen?
2 NH3 → {Glutamine synthetase} → Glutamine
There is a symbiotic association between bacteria and yeast in the soil (associated with the roots) → Bacterias fix Nitrogen (Nitrogenase catalyses N2 → 2 NH3, ATP-dependent)
NH3 is taken up by plants which assimilate it in their cells (Many different nitrogen assimilation reactions for ex: Glutamine synthetase catalyses NH3 → Glutamine)
What are the 4 most important nitrogen assimilation reactions?
a-ketoglutarate (0N) → {Glutamate dehydrogenase} → Glutamate (1N) (→ other AA)
Glutamate (1N) → {Glutamine synthetase} → Glutamine (2N) (→ Purine nucleotides, AA, tryptophan, histidine)
Aspartate (1N)} → {Asparagine synthetase} → Asparagine (2N)
CO2, ATP → {Carbamoyl phosphate synthetase} → Carbamoyl phosphate (→ Arginine, pyrimidine, urea)
What is the role of pepsin?
- Secreted in the stomach
- 1st protein digestion enzyme → polypeptides
- Activated at pH ~2
- Cleaves after Phe, Leu, Trp, Tyr
What is the role of trypsin?
- 2nd protein digestion enzyme of GI tract (polypeptides → peptides)
- Secreted by the pancreas into the duodenum
- Cleaves after Arg, Lys
What is the role of chymotrypsin?
- 3rd protein digestion enzyme (polypeptides → peptides)
- Secreted by the pancrease into the duodenum
- Activated by trypsin
- Cleaves after Phe, Trp Tyr
What is the role of Aminopeptidase and Carboxypeptidase A?
- 4th protein of digestion enzyme (peptides → amino acids)
- Secreted by the pancrease into the small intestine (taken up by the brush border)
- Non-specific cleavage → Aminopeptidase cleaves of the N-terminus / Carboxypeptidase cleaves of the C-terminus
How does uptake of AA into enterocytes work? What is the exception?
20 AA with different combinations of dipeptides and tripeptides uptaken → many many specific transporters at the brush border of enterocytes
General rule → imported via Na+ and H+ symporters
Exception in neonatal mammals:
Whole milk proteins are endocytosed and degraded in lysosome (AA go into circulation) or pass unscathed through the enterocyte to circulation
What are essential, non-essential and conditionnally essential amino acids?
Essential → AA you can’t synthesize, you need to ingest in diet
Non-essential → AA you can synthesize (ex: Alanine, Asparagine, Aspartate, Glutamate, Serine)
Conditionnally essential → requires essential AA as a strating material to synthesize them (ex: Cysteine requires methionine and Tyrosine requires Phenylalanine)
What are the different cellular uses of the amino acid pool?
- Energy production
- Synthesis of gluocse or fatty acids
- Synthesis of nonprotein molecules that contain nitrogen
*in cells, only have the L enantiomer (side-chain on the left, COO- down and NH2 up)
What are the different principles of the amino acid pool?
- Homogenous → no matter the source (diet or endogenous/ vegetable or animal), alanine is alanine for ex
- Dynamic (constant breakdown and synthesis)
- Constant size → Body protein (~10kg of dry mass) + free AA pool (~0.1kg) in a 70 kg adult)
- All AA must be aailable in the pool at the same time for protein synthesis
*The body can’t store N-containing molecules as it does for carbohydrates and lipids → animals must replenish nitrogen supplies through the diet to replace nitrogen lost through catabolism
What is the effect positive and negative AA balance?
Equilibrium: Intake = Outflow
Positive balance: Intake > Outflow → growth, pregnancy, weight lifting, etc.
Negative balance: Intake < Outflow → Illness, surgery trauma, cancer cachexia
Where is the carbon skeleton of AA recycled?
- To glucose
- TCA intermediates
*Must be deaminated: AA → carbon backbone + NH3 (toxic)
What is considered to be a high [amminium]? What is the effect of high [NH3]?
normal blood [ammonium] < 50mM
high concentration → Disturbance of consciousness, coma, convulsion, death
Found in the blood as ammonium (NH4+): NH3 + H+ + OH- ←→ NH4+ + OH-
*Still must be detoxified → Urea cycle (in the liver) → Urea is excreted in urine which is then degrade by bacteria and they regenerate N2
Which are the 2 most abundant AA?
- Alanine
- Glutamine → key AA for protein synthesis, N-compounds, non-toxic storage vehicle for NH4+ (Glutamate + NH3 → Glutamine)
What is the importance/role of Glutamine synthtase and Glutaminase?
They regulate the pool of glutamine (both in the peripheral tissues and in the liver)
*Glutamine is the circulating form in the blood as it carries a NH3
Glutamine → {glutaminase releases NH3} → Glutamate → {glutamate dehydrogenase release NH3} → aKG
Where in the body are the following enzymes found?
- Transaminases
- Glutamine synthtases
- Glutaminase
- Glutamine dehydrogenase
Transaminase and Glutamine synthetase → peripheral tissues (including muscles for example)
Glutaminase and Glutamine dehydrogenase → release NH3 → in the liver only (→ urea cycle)
Which is the 1st enzyme responsible for detoxifying NH3 during exercise?
Exercise → AA breakdown for energy production (fill CAC) → release of NH3
In the peripheral tissues, GLUTAMINE SYNTHETASE puts the free NH3 onto Glutamate → to make Glutamine which will act as a carrier for the NH3 through circulation to the liver → urea cycle
What reaction is catalysed by Glutamine synthetase
Glutamate (OH-C(R)=O) → y-Glutamyl phosphate (consumes 1 ATP) → Glutamine (H2N-C(R)=O)
*NH3 comes from AA breakdown
*Phosphate acts as a high-energy intermediate to help generating the C-N bond
*In peripheral tissues
What reaction is catalysed by Glutaminase?
*In the liver only
Glutamine → {uses H2O, releases NH4+} → Glutamate
*Opposit reaction as Glutamin Synthetase
*NH4+ goes to the urea cycle
What is the difference between a synthase and a synthetase?
Synthetase → uses ATP
Synthase → does not use ATP
What is a deamination reaction?
Give 2 examples of enzymes.
It removes the nitrogen group from a molecule and releases it as NH4+
Enzyme IN THE LIVER:
- Glutaminase
- Glutamate dehydrogenase
*NH4 is released to the urea cycle so has to be in the liver
What drives the Glutaminase reaction forward?
Consumption of NH4 by the urea cycle → net concentration gradient between products and substrates
*Almost only in the liver (minor in the kidney)
What are key features of the Glutamate dehydrogenase deamination?
Glutamate → a-ketoglutarate + NH4+
- In the mitochondrial matrix of the kidney, liver and nevous tissues
- Uses NAD+ or NADP+ as an electron acceptor/co-enzyme
∆G˚’ = 30kJ/mol → endergonic
*Consumption of NH4 by the urea cycle + consumption of aKG by the CAC drives the reaction forward in vivo (would not occur in a petri dish)
What regulates glutamate dehydrogenase?
Regulated by the energy state:
- Low ADP (and NAD(P)) = allosteric activator
- High GTP (and NAD(P)H) = allosteric inhibitor
What is the reverse reaction to Glutamate dehydrogenase?
*Same enzyme
Glutamate synthesis:
a-Ketoglutarate + NH4+ + NAD(P)H → Glutamate + H2O + NAD(P)+
What is different in the nitrogen metabolism of cancer cells?
Glutamate, Glutamine and NH3 in circulation sent for disposal are taken up as a source of energy by cancer cells
What are transamination pairs?
Give examples.
Amino acid (-NH3) and its keto acid (=O)
Glutamate - a-Ketoglutarate
Aspartate - Oxaloacetate
Alanine - Pyruvate
*Both can be ana/cataplerotic transamination reactions
What if the required co-factor for transamination reactions?
Pyridoxal phosphate
What are important features of Pyridoxal-5’-Phosphate (PLP)
- Derived from Vitamin B6 (found in food)
- Involved in many different reactions → transaminations, decraboxylations, racemizations, eliminations, aldolizations
C 4’ aldehyde group → will bind covalently to a-amino groupe of enzyme residue/substrate
Phosphate group → called the “handle” → for binding to the PLP coenzyme
3-OH group improves catalytic efficiency
Pyrimidine ring → electron sink involved in resonance for stabilization of carbanion intermediates
