bmsc 230 module 11 protein degradation and urea cycle

glucogenic amino acid

an amino acid that can be converted into glucose through gluconeogenesis. (can be broken down into an intermediate of glycolysis or TCA cycle)

ketogenic amino acid

an amino acid that can be converted to ketone bodies. (degraded to acetyl CoA or acetoacetate)

phenylketonuria

A genetic disease resulting in

• decreased metabolism of phenylalanine a

• build-up of harmful side-products of an alternative pathway for phenylalanine degradation.

Porphyrias

Rare genetic diseases that lead to defects in the biosynthesis of porphyrins and accumulation of intermediates of the synthetic pathway.

Transdeamidation

the combination of amino acid transamination and oxidative deamination of glutamate.

Ureotelic organism

Organisms whose main way to excrete excess nitrogen is via urea

Uricotelic Organism

Organisms whose main way to excrete excess nitrogen is via uric acid

Zymogen

An inactive enzyme that is converted into its active form when part of it is cleaved from it by a proteolytic enzyme.

the build up of what in our bodies is toxic?

amino groups (in the form of ammonium, NH4) from amino acids

is there a storage depot for amino acids?

no

in what cases will muscles be degraded to provide amino acids for the body to use for things like fuel, or formation of other proteins?

severe metabolic or physiological disorders such as starvation or untreated diabetes

what are the sources of amino acid input into our bodies?

dietary proteins, turnover/degradation of cellular proteins, synthesis of amino acids by the cell

can mammals synthesize all 20 amino acids?

no. only some yeasts and bacteria can.

since mammals cannot synthesize all 20 amino acids, where do the rest of them come from?

diet

what are amino acids from the amino acid pool used for?

• to make new proteins

• synthesize other nitrogenous compounds

• metabolized by different pathways to biomolecules such as glucose, ketone bodies, and fatty acids,

• oxidized through the citric acid cycle to CO₂ and H₂O, producing ATP in the process

organisms need to be in _____ due to the toxicity of excess ______

nitrogen balance, ammonium

can intact proteins be absorbed into the small intestine?

no, they are too large

what does digestion do?

breaks down proteins into primarily amino acids, as well as a small amount of di- and tri-peptides.

what is proteolysis?

The process whereby peptide bonds within proteins are cleaved

• this is a hydrolytic reaction where a water molecule is added across a peptide bond

where does proteolysis start?

stomach

where is protein digestion completed?

in the small intestine, through proteolysis

how does chewing facilitate digestion?

liquefies protein

• increases its surface area

• makes it a better substrate for the proteases it will encounter in the stomach in small intestine.

chewing and having the protein in your mouth triggers neural effects

• prepares the stomach for the protein that is on its way

what do cells in the stomach secrete in response to neural effects of chewing?

gastric juice

what is the pH of gastric juice?

2

what other ingredients are found in gastric juice?

gastrin and pepsin

what purposes does the low pH of gastric juice serve?

1. causes denaturation, or the unfolding, of proteins.

   • facilitates the degradation of proteins, since unfolding of proteins makes the peptide bonds accessible to proteases
     

2. antiseptic

   • destroyins many bacteria and viruses that are ingested when we eat.

what is gastrin?

• a peptide hormone

• secreted by special cells in the stomach.

• primary role is to stimulate acid secretion into the stomach.

what is pepsin?

• a protease that cleaves the peptide bonds involving hydrophobic amino acids.

• doesn’t completely digest a protein

• breaks it up into smaller segments which facilitates the next round of proteolysis that occurs in the small intestine.

what does pancreatic juice do?

• secreted into small intestine

• neutralizes digestive slurry between stomach and small intestine so that the cells lining the small intestine aren’t damaged and so that the digestive enzymes present in the small intestine aren’t denatured.

• contains bicarbonate, which neutralizes the gastric juice to around a pH of 7.0.

• also contains several additional proteases, each recognizing different types of peptide bonds

what is trypsin?

a protease that cleaves bonds following arginine or lysine

what is chymotrypsin?

a protease that cleaves bonds following aromatic amino acids

what is elastase?

a protease that cleaves bonds after amino acids that have smaller, hydrophobic side chains

what do carboxypeptidases and aminopeptidases do?

cleave bonds working from either end of peptides with little specificity.

what absorbs the amino acids that are cleaved off of proteins using proteases?

absorbed by the endothelial cells and then into the portal vein, where they are transported throughout the body for utilization.

are proteolytic enzymes always active?

no. they are stored in granules after they are synthesized, and then released from these granules when needed.

how are the proteases enzymes themselves protected from being degraded by their own proteolytic activity?

they are initially synthesized in an inactive form called a zymogen, and are activated by having a small portion of their polypeptide backbone cut off which allows them to properly fold into active enzymes.

what is the zymogen of pepsin called?

pepsinogen

What triggers the removal of the masking sequence in proteolases?

• the low pH of gastric juice causes a small degree of conformational change

• allows it to self-cleave the masking sequence, forming a small number of active molecules.

• These active molecules in turn attack other inactive proteolases, converting them to active molecules.

• This self-activation process is called autocatalysis.

how is protease activity is terminated when not needed?

they self-inactiavate

what is GERD?

result from an over-active proton pump in the stomach, resulting in the production of too much acid.

• Normally, the K⁺/H⁺ pump is kept sequestered in intracellular membrane compartments until it is needed at the cell membrane.

• Signalling molecules such as gastrin and histamine bind to receptors on parietal cells,

• leads to activation of protein kinase A by increasing cAMP in the cell.

• phosphorylates the K⁺/H⁺ pump,

• begins pumping protons into the lumen of the stomach, lowering the pH.

  this signalling pathway becomes dysregulated, resulting in too much acid secretion which lead to heartburn.

what types of medications exist to treat GERD?

• Antacids such as TUMS (calcium carbonate) act as sponges for protons, thus reducing acidity.

• medication that binds to and inhibits the histamine H2 receptor by preventing the binding of histamine, which puts the brake on the cAMP signalling pathway and slows the translocation of the proton pump to the cell surface.

• Proton Pump Inhibitors (PPIs) bind directly to the proton pump and inhibit its ability to pump protons into the lumen of the stomach.

what are the two components of an amino acid that we consider?

amino groups and carbon skeletons

how are amino groups excreted from the body?

in the form of urea

what happens to the carbon skeletons?

• converted into alpha-keto acids, which can be oxidized for energy production

• or in some cases converted to glucose via gluconeogenesis.

the amino groups are funneled into what?

glutamate

the amino group from glutamate is released as what?

ammonium (NH4+)

what is ammonium used for?

to synthesize urea, which is excreted from the body

where in the body does urea production occur?

only in the liver

what is the first step in amino acid degradation?

transamination, which gathers the amino groups from 17 amino acids into the amino acid glutamate

which 3 amino acids are excluded from transamination?

glutamate, lysine, and threonine

what type of enzyme catalyzes transamination?

aminotransferases, which are specific for the amino group donor.

All 17 of the aminotransferases transfer the amino group from the amino acid substrate to____, converting it to ______.

alpha-ketoglutarate, glutamate

aminotransferases require _____, which is derived from vitamin _____, as a coenzyme.

pyridoxal phosphate, B6

are aminotransferases reversible enzymes?

yes. even if and when this reaction proceeded in the reverse reaction, the name of the enzyme doesn’t change

what is alanine aminotransferase?

the enzyme that transfers the amino group from alanine to alpha-ketoglutarate

what are the products of alanine aminotransferase?

glutamate and pyruvate

what is aspartate aminotransferase?

the enzyme that transfers the amino group from aspartate to -ketoglutarate

what are the products of aspartate aminotransferase?

glutamate and oxaloacetate

where does transamination occur?

cytosol

what happens after transamination?

oxidative deamination of glutamate

where does oxidative deamination of glutamate occur?

mitochondria of liver cells. The glutatmate moves into the mitochondria from the cytosol.

Glutamate undergoes oxidative deamination, which releases the amino group as ___ and _____

ammonium (NH₄⁺), alpha-ketoglutarate.

where is free ammonium produced?

in the mitochondria, rather than cytosol, because this is where it is used to form urea

what enzyme catalyzes oxidative deamination of glutamate?

glutamate dehydrogenase. this enzyme catalyzes the removal of the amino group, as well as the oxidation of glutamate in a two-step process

what is the substrate of glutamate dehydrogenase?

can use either NAD⁺ or NADP⁺

In most tissues (excluding muscle), free ammonium is incorporated into glutamine by the enzyme ___

glutamine synthetase

Can glutamine get out of cells and into the blood stream?

yes. it is transported to liver via bloodstream

what enzyme, in the liver, uses water to cleave off ammonium from glutamine, producing glutamate and free ammonium?

glutaminase

in most peripheral tissues, after glutamate is produced from glutamine, how is the second amino group released?

The glutamate is acted upon by glutamate dehydrogenase

what amino acid transports ammonium out of muscle tissue?

Alanine.

Initially, the excess amino groups in muscle are collected in the form of ____, which then undergoes a transamination reaction with ____,

glutamate, pyruvate

the reaction of glutamate with pyruvate is catalyzed by what enzyme? What does it form?

alanine aminotransferase, which transfers the amino group from glutamate to pyruvate, forming alanine

Alanine leaves the muscle, goes to the liver through the blood, and in liver the ____ present there transfers the amino group from alanine to ______, reforming ______.

alanine aminotransferase, alpha-ketoglutarate, glutamate

the glutamate, in the liver, that is reformed from alanine, in muscle tissues, is acted upon by which enzyme? What is released?

glutamate dehydrogenase in the mitochondria; this releases ammonium which is converted to urea. Pyruvate is also released, and is used for gluconeogenesis to make glucose (which is then metabolized through glycolysis to pyruvate: glucose alanine cycle)

is urea water soluble?

yes

why does urine have an odor?

because urea breaks down, in an uncatalyzed or spontaneous fashion, into ammonia

where in the cell do the reactions of the urea cycle take place?

mitochondira and cytosol

what part of the cell does the urea cycle start in? why?

the mitochondria, because ammonium is released in the mitochondria

what is the first and rate-limiting step of the urea cycle?

ammonium is fused to CO₂ and requires the cleavage of two ATP, forming carbamoyl phosphate.

• This step is where free ammonium is used in urea synthesis.

• catalyzed by carbamoyl phosphate synthetase I.

what is the obligate allosteric activator of carbamoyl phosphate synthetase I?

N-acetylglutamate

N-acetylglutamate is synthesized from glutamate and acetyl CoA by the enzyme _____. This enzyme is stimulated by the presence of _____

N-acetylglutamate synthase, arginine

carbamoyl phosphate combines with _____to produce _____in the mitochondria. The enzyme that catalyzes this is _______

ornithine, citrulline, ornithine transcarbamoylase

After citrulline is made in the mitochondria, where does it go to?

cytosol

how many amino groups does urea have? Where do they come from?

Two. one comes from free ammonium. The other one comes from aspartate

what is the source of the amino group in aspartate?

glutamate

glutamate provides amino groups used in urea synthesis in which two ways?

1. free ammonium is cleaved from glutamate by glutamate dehydrogenase, which is used to form carbamoyl phosphate.

2. glutamate transfers its amino group to form aspartate, which is used later in the urea cycle to add the second amino group onto urea.

citrulline reacts with ____to form ____.

aspartate, argininosuccinate

citrulline is acted upon by ___

argininosuccinate synthetase.

argininosuccinate synthetase uses what to drive the reaction forward?

ATP

Argininosuccinate is then acted on by _____, which cleaves the substrate into ____and ____

argininosuccinase, fumarate, arginine

the side chain of arginine has how many amino groups?

2 (one came from the free ammonium used to synthesize carbamoyl phosphate, and the other came from aspartate)

when arginine is acted upon by the hydrolase ____, ____is produced along with _____

arginase, urea, ornithine

what happens to the ornithine that is made from hydrolyzing arginine?

it re-enters the mitochondria and reacts with another molecule of carbamoyl phosphate and starts another round of the cycle.

the three cytosolic enzymes (argininosuccinate synthetase, argininosuccinase, and arginase) are ______

clustered together in a complex

why are argininosuccinate synthetase, argininosuccinase, and arginase clustered together in a complex?

• it keeps the pathway intermediates trapped in or near the complex. This keeps the local concentration of intermediates high, thus increasing the reaction rate of the cycle.

• prevents the intermediates from undergoing unrelated, side reactions which would slow the rate of flux through the urea cycle and inhibit the elimination of excess nitrogen from the cell.

what happens to the fumarate that is cleaved from argininosuccinate?

it can either go directly to the citric acid cycle or be first converted to malate

when glutamate transfers its amino group to oxaloacetate, what is formed?

aspartate

aspartate can form ___ in the cytosol, to provide one of the amino groups present in urea

argininosuccinate

urea synthesis is connected to the citric acid cycle through ____

fumarate and aspartate

how is urea synthesis regulated?

through N acetylglutamate and arginine