IMED1003 - The Urea Cycle (L20)

Summary so Far

- the name aminotransferase is exactly how it sounds. It is the transfer of amino group from one substance to another.

- another name for glutamate is glutamic acid (which is why you dont want it in the blood, can cause acidosis)

Why Ammonia is toxic

- tolerated conc of ammonia in blood is around 50 micromoles per litre

- neurlogical issues ensue above this

- these include ataxia, and if ammonia isn't cleared from blood you can go into a coma

What u need to know about Urea Cycle

- dont need to do all ractions, but you need to know the ATP requirements becuase this is a vital pathway we undertake

- also need to know the overall reaction

- some of the key enzymes

- where the enzymes are active

NH3 made in tissues is toxic

- must maintain low blood [NH3]

- two "ways" to move N safely in blood: Gln or Ala

.

- the reason we use these ones is because they are uncharged and will not affect tonicity of sol

- muscles make alanine, most other tissues make glutamine

- these amino acids can then travel in the blood and go to the liver safely

- glutamate differs from glutamine because glutamine has an extra amino group

How the muscles make use of alanine

- the muscles make alanine and shunt it through blood to the liver

- in the liver alanine will have amino group removed so it becomes pyruvate

- pyruvate is glucogenic and hence the liver can make glucose from it

- the muscles then absorb that glucose thats produced

Enzymes for amine movement

- Alanine to Pyruvate: alanine amino transferase (ALT) (also reversible)

- Glutamate to glutamine: glutamine synthase (Gln synthase)

Glutamine

- made in many tissues, NH3 transferred to Glu by Gln synthase -> Gln --> blood

- in liver, glutaminase removes NH3 to reform Glu - can be processed to alpha-ketoglutarate

- Then ALT Ala -> Pyruvate, used in gluconeogenesis

.

- glutamine synthase is expressed in basically all tissue. the conversion to glutamine requires ATP, but now glutamine can be safely transported to liver

- glutaminase reverses the above reaction, stripping off the amino group and forming glutamate

- basically we have safely moved an amino group from the body cells to the liver, where now the NH3 can form urea

What the liver can do with leftover glutamate

- glutamate is now present after the NH3 was removed from glutamine by glutaminase

- liver has high levels of Glutamate Dehydrogenase which means we can strip off the second amino group from glutamate

- the second amino group could have come from when another amino acid added amino group to alpha-ketoglutarate to form glutamate

Alanine Delivery

- the alpha-ketoglutarate and glutamate pairing, combined with alanine in the presence of ALT can form glucose

Transport of NH3 to liver EQUATIONS

DIAGRAM ON SLIDE 8

Alanine Amino Transfer to Liver

- in muscle, AA transamination to Glu. ALT produces Ala from pyruvate and Glu

- Ala --> blood

- in liver, ALT reverses this to give Glu and pyruvate - use in gluconeogenesis and return glucose to muscle = glucose-alanine cycle (NOT THE CORI CYCLE)

Nitrogen Excretion

- urea cycle - liver and kidney

- Cylical, so reutilise intermediates

- Major route for removal of ammonia - AA catabolism

- NH3 toxic - need low blood [NH3], transported from tissues to liver as Gln and Ala

- Urea - soluble, excreted in urine.

- Not all urea excreted, lots processed in gut by bacteria

- Urea N - from NH3 and Asp

Urea Cycle Components

- ornithine is the starting point

- ornithine is derived from arginine and functions in urea cycle as a carrier of nitrogen

- citrulline is another different type of amino acid that is a nitrogen carrier

- Carbamoyl phosphate synthase 1 makes carbamoyl phosphate by combining CO2 and NH4+ or bicarbonate and ammonia and uses 2 ATP to synthesise the activated carrier of amino group

- ornithine then binds to carbamoyl phosphate in the mitochondria and forms citrulline

- citrulline moves back out into cytosol and combines with aspartate and our second amino group of urea using 2 ATP equivalents to form argininosuccinate

- from argininosuccinate we produce arginine and we release 4 carbon fumarate

- from fumarate we can get OAA (TCA Cycle)

- from arginine we can reform ornithine

ALL PAIRS U NEED TO KNOW SO FAR

- Alaine and Pyruvate

- Aspartate and OAA

- Glutamate and Alpha-Ketoglutarate

- IN THE UREA CYCLE: Aspartate and Fumarate

UREA CYCLE + TRANSPORT DIAGRAM

- glutamate cant travel in the blood becuase its acidic which is why it travels as glutamine

- glutamate dehydrogenase releases amino group

Making Urea - 1N from NH3

- Glutamate is a principal amine donor to other AA

- Free ammonia released from glutamate is converted to urea for excretion

- Carbamoyl phosphate synthase 1 captures free ammonia - mitochondrial matrix

- First step (nitrogen-acquisition) of urea cycle, requires 2ATP, multistep

DONT NEED TO KNOW THE DETAILS

Making Urea - 1N from Asp

- Second nitrogen-acquiring reaction - cytosol

- Enzyme = argininosuccinate synthase, uses 2ATP equivalents, 2 step process

- Citrulline's O is activated using ATP, then aspartate added and AMP displaced, making argininosuccinate

FULL UREA CYCLE DIAGRAM

- enzyme you have to remember is called ornithine transcarbamoylamylase (because it adds carbamoyl group to ornithine)

- this is the most common issue that people have with their urea cycle is that they either dont make enough or its not functioning well enough

- the enzyme that cleaves argininosuccinate to arginine is called argininosuccinase

SIMPLIFIED REACTIONS OF UREA CYCLE

DIAGRAM ON SLIDE 17

Overall Reactions of Urea Cycle

NH4+ + HCO3- + aspartate + 3ATP + H2O --> urea + fumarate + 2ADP + 2Pi + AMP + PPi

- equivalent of 4 ATP used

- NEED TO RMBR OVERALL RXN

Regulation of Urea Cycle

- expression of urea cycle enzymes increases when needed, usually enough enzymes to cope with a reasonable amount of AA catabolism, but need more if:

- High protein diet or starvation - protein being broken down for energy

- get upregulation of enzyme gene transcription and translation

Regulation of Urea Cycle through chemicals

- Allosteric regulation of carbamyol phosphate synthase 1 (CPS1)

- activated by N-acetylglutamate (precursor of ornithine)

- so, after a high protein meal, get activation of enzymes for urea production and supply of glutamate to make the activator or CPS1

aspartate-argininosuccinate shunt

NH4+ + HCO3- + aspartate + 3ATP + H2O --> urea + fumarate + 2ADP + 2Pi + AMP + PPi

- there is communication between the amino and carbon skeleton part

Aspartate-Argininosuccinate Shunt Links Urea Cycle and Citric Acid Cycle

DIAGRAM ON SLIDE 22

SUMMARY

DIAGRAM ON SLIDE 23