Exam 2

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

AA1 +a-ketoacid2

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

Alanine and a-Kg

Glutamate oxaloacetate transaminase

Glutamate and oxaloacetate

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

-Sum of transaminase reaction and GDH reaction
AA1+NAD+H2O

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

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

catalyzes deamination of Histidine
His

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