Studied by 19 people
Primary Metabolites
needed for normal operation of metabolic pathways and main cellular functions, such as AA, DNA, RNA
Secondary Metabolites
not needed for life. Important for drugs and plants. not needed for cell growth, development or reproduction. Ward off pathogens and predators, protect against osmotic damage.
Essential Amino Acids
Phe, Val, Thr, Trp, Ile, Met, His, Leu, Lys
Arginine
conditional AA during growth
Tyrosine
condition AA if Phe is low
Cysteine
conditional AA if Met is low
de Novo Synthesis
have ways to make gultamate and other non essential amino acids. Can break down to make amino acids if too much metabolic stress. Each organ has a different metabolism
Muscles
primarily hydrophobic AA because they get heated quickly
Liver and Brain
Liver makes a lot of AA, Brain makes lots of Q
Protein Fragments
Created after proteolysis in stomach due to low pH. Low pH protonates all proteins, have either positive or neutral charge which causes proteins to repel each other and open up. Now easy to cut.
Saliva
Mainly contains proteases from bacteria and WBC, not much going on.
Pepsin
in stomach; cleaves after Phe, Leu, and Glu linkages. ACRONYM: ELF
Chymotrypsin
in intestine; cleave on carboxy side of peptide bond of aromatic residues
Trypsin
in intestine; cleaves on carboxy side of lysine and arginine residues
Carboxypeptidase
in intestine; cleaves C-terminal AA
Elastase
in intestine; cleaves elastin
Why can't protein fragments refold in small intestine, which has a neutral pH?
Protein fragments all cut up from Pepsin, cannot refold
When are proteins proteolyzed into AA or Di/Tri-peptides?
After the small intestine
Are dietary proteins absorbed in intestine?
No, proteins are seen as foreign bodies. You do not want the body to overreact, so proteins must be proteolyzed to AA, Di/Tri peptides. Larger structures are not important in circulation
Zymogens
inactive form of proteases. Stored and made in pancreas. Activated after secretion into small intestine to protect pancreas. Prevents autophagy and apoptosis
Trypsinogen
zymogen of trypsin. Made in pancreas and stored in vesicle with trypsin inhibitor
Trypsin Inhibitor
Prevents unwanted proteolysis of host cells
Enterokinase
ectoprotease on intestinal mucosal wall that converts the zymogen to active form of trypsin
Pro-carboxypeptidase
activated to form carboxypeptidase, which removes AAs one by one from C-terminus of food protein
How would proteases react without carbohydrates?
Protease would bind to other protease thinking it is a protein substrate, causes health issues
Zymogen Activation
Zymogen synthesis, processing and transport in ribosome of rough ER, go to golgi for vesicle targeting and release
Pepsinogen
autocalalytic, meaning once pepsin forms, it catalyzes cleavage of pepsinogen to make more pepsin. Slow activation by stomach
Aspartic Proteinase
pepsin operates at low pH by using its aspartic acid carboxyl groups for catalysis
Gastric Chief Cells
pepsinogen is formed and released in these cells within stomach
Intracellular Protein Turnover
When nitrogen intake is low, protein degradation is high because cells need amino acids to make essential proteins. Two paths, either lysosomal or ubiquitin pathway
Lysosomal/Phagolysosomal Pathway
Lysosome is acidic, proteins undergo partial unfolding via isoelectric expansion. This makes proteins more susceptible to proteolysis. ATP-dependent pump H+ into this compartment to make this proton gradient.
Ubiquitin-dependent Pathway
After chaperonins have failed to help guide protein folding, ubiquitin is enzymatically joined to misfolded protein. Ubiquitin is ticket to get into proteasomes, which is a barrel-like protease complex. Gets stuck and degraded.
Intestinal Lumen
High Na+, and AA
Positive Nitrogen Balance
growth, healing of wounds, convalescence intake>excretion
Negative Nitrogen Balance
starvation, malnutrition, disease excretion>intake
Marasmus
Not enough protein of kcals causing extensive tissue and muscle wasting with no edema. hair stays for little warmth
Kwashiorkor
Enough kcal but not enough protein in diet. Causes irritability, enlarged liver due to fatty infiltrates from high carb diet, and edema caused by hypoalbuminemia.
PLP
has an aldehyde group
PMP
has a primary amino group which preserves the energy from the peptide bond of amino acid 1 in the first half reaction
Transaminase Reaction Sum
Transaminase: First half reaction
Form aldimine (aldehyde but instead carbonyl have N bonded to CH), which gets converted to ketimine and hydrolyzes to ketoacid
Transaminase: Second half reaction
Ketimine goes to aldimine. Just the reverse of reaction 1 but using R2-KA to make R2-AA.
Transamination
has the same bonding in products as in substrate, hence a Keq of 1. Transpose the ketone and the amino group to opposing molecules.
Ala a-kg transaminase
Glutamate oxaloacetate transaminase
Which amino acids cannot undergo transamination?
Proline, hydroxyproline because too many carbon bonds due to secondary amines Lysine and Threonine because they would form toxic metabolites
Deamination
Regenerates a-Ketoglutarate as an amino acceptor and provides ammonia for either reutilization or disposal as urea
GDH
located in the mitochondria, master control Uses Glutamate, NAD+, and H2O to make a-Kg and NH3
NAD+
oxidized form
NADH
reduced form
NADH oxidation
makes 3 ATP
NADH and NADPH
allow cells to manage chemical energy, can help to reduce other molecule
Does the mitochondria efficiently oxidize NADH?
Oxidized so efficiently that nothing is around, mainly NAD+ in the mitochondria for GDH to use.
What amino acids can't be deaminated oxidatively through the coupling of GDH with transaminases?
Pro, Hyp, Thr, Lys, His
Oxidative Deamination
When does NADH get reoxidized into NAD+?
Oxidative phosphorylation in the mitochondria
Where does excess NH4 from oxidative deamination go?
Urea cycle
Why is GDH a master control enzyme? Why can't we have an enzyme for each amino acid?
All enzymes would have to be like GDH. A mutation would cause death due to nitrogen accumulation being toxic
How does GDH catalyze oxidation?
NAD+ levels are really high due to NADH oxidation making ATP in mitochondria. ONLY FORWARD Therefore, there is... Excess NAD+ + Glu -> a-Kg+NH4 + low NADH
How does GDH catalyze reduction?
Other pathways generate tons of NADPH to make fatty acids, sterols, and more. ONLY REVERSE. Therefore, there is... low NADP+ + Glu <- a-kg+NH4+excess NADPH
How does High ATP, GTP, and NADH affect GDH activity?
Inhibits GDH since this favors protein synthesis (use ATP to make peptide bonds), reduces AA degradation. Not oxidizing AA
How does High ADP, GDP, as well as excess AAs affect GDH activity?
Activate GDH. These conditions indicate there is little energy in the body. a-kg stimulates Krebs Cycle, which fuels ETS and Oxidative Phosphorylation.
What pH favors deamination?
A low pH
What pH favors ammination?
High pH. Think basic bonditions, want to be ammonia and not a ketone
Why do we use the L- and D- amino acid oxidases infrequently?
Enzyme is not regulated and produces H2O2, which is highly toxic to body.
Why do we need both L and D AA oxidases?
Don't want to waste energy bonds in D-AA. Protein ages, forms D isomer. Also has a antibacterial affect since D isomers are found in bacterial wall.
Overall reaction of L-D AA Oxidases
AA+O2-> a-ketoacid +NH4++H2O2, uses FMN
Glutaminase
hydrolysis of glutamine to deaminate. Occurs in mitochondira to not interfere with glutamine synthetase. Makes ammonia that is sent to urea cycle Q+H2O-> E+NH3
Asparaginase
catalyzes hydrolysis of Asn, deamination Asn+H2O->Asp+NH3
Histinase
Ammonia Assimilation
metabolically incorporate ammonia to keep concentration low since high concentrations are toxic
How is ammonia toxic to humans?
NH3 can easily pass membrane because it is neutral and steal protons that are there to maintain proton gradients. They can also deplete a-Kg because NH3 can drive the GDH reaction, which is needed for the Krebs Cycle which can interfere with ATP production.
Which enzymes, relevant to this course, are used for Nitrogen assimilation?
GDH (reverse rxn with NADPH), Glutamine Synthetase, and CPS-1
Where is Glutamine Synthetase located?
In the cytosol to prevent a futile cycle with GDH
Glutamine Synthetase Rxn
ATP+NH3+Glutamate->Glutamine+Pi+ADP+H+
What is the main way to trap NH3?
Glutamine synthetase
How to avoid direct transfer of NH3 from distant organs to the liver?
Shuttle it around as Gln, never want to have ammonia in the circulation to prevent inducing a coma, protect the brain
What is the major nitrogen source?
Glutamine
Glutamine Synthetase and ∆G ATP hydrolysis
phosphorylates negative O in E to make a better leaving group and drive ammonium ion deprotonation at neutral pH
How many SN2 displacement reaction in Glumatine synthetase?
1 to add a phosphate to make a better leaving group and 1 to use NH3 as a nuc to attack carbonyl
How do we deprotonate NH4 when we are at neutral pH and the pKa of NH3 is 9.2?
Use ∆G from ATP hydrolysis to deprotonate, and use arginine, with a pKa of 12, to manipulate the pH near NH4 to deprotonate right before the reaction
What is the essential intermediate of Glutamine Synthetase?
∂-glutamyl-P, which is just a phosphorylated glutamate
Where is CPS-1 located?
Mitochondria, need it to be there to make it for the urea cycle (for ornithine)
CPS-1 rxn
2 ATP+NH3+HCO3--> 2HN(C=O)-O-P+ Pi+2ADP+H+
CPS-1 mechanism
Phosphorylate bicarb in step one to form carboxyl P as the intermediate which immediately gets attaked by NH3 to kick P off. Carbamate gets phosphoryalted again into final product
Why is glutamine vital for the efficient biosynthesis of nitrogen containing metabolites?
Allows glutamine dependent biosynthetic enzymes to generate ammonia in active site. That ammonia is then used as a nuclephile, without ever touching water so it cannot get protonated.Transfer ammonia to another nuc, will never touch water and won't go back to ammonia
Where is CPS-2 located?
Cytoplasm, site of pyrimidine nucleotide synthesis
Benefits of CPS-2
Hydrolyzing glutamine to deprotonate NH3 exactly where and when it is needed, and avoids toxicity of ammonia elsewhere in our cells
How is water kept out of the tunnel for CPS-2?
Molecular weight of ammonia is lower than that of water. Also, water always bundles up to other water molecules unless at 100C, and a single one can't get into skinny tunnel. Tunnel is also hydrophobic.
Glutamine-Hydrolyzing subunit
Hydrolyzes Gln to Glu+NH3. The essential -SH group generates a y-glutamyl thiolester that occupies the active site, permitting NH3 to transfer quicker.
Biosynthetic subunit
Unique to particular biosynthetic enzyme
What is the lid of the Q site?
Covalently bound E
What is the activator for CPS-1?
N-Acetyl-Glutamate
For what does CPS-1 have a high affinity?
NH3
Ammonotelic
NH3-excreting, fish just swim away
Uricotelic
Uric acid secreting, like birds. Highly oxidized
Ureotelic
Urea excreting
Gout
Too much uric acid
Where is the principal site of NH3 detoxification?
Liver
In what organic forms is ammonia transported in?
Urea, Glu, Gln, Ala
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