1 - Urea

Urea Cycle Steps

1) Carbamoyl Phosphate + Orthinine --> Citruline (Citruline leaves mitochondria and enters cytosol) 2) Citruline + ATP (adds AMP) ---> Citrullyl-AMP Intermediate +PPi 3) Citrullyl-AMP Intermediate + ASPARTATE ---> Arginosuccinate 4) Arginosuccinate --> Fumarate + Arginine 5) Arginine releases Urea ---> produces Orthinine 6) Orthinine enters mitochondria to be reused in another cycle

Reversible Transamination of Asp <-> Glu (via Aspartate Aminotransferase)

Aspartate Aminotransferase Enzyme reaction Example of a Transaminase reaction: 1) Aspartate donates its amino group, becoming the a-keto acid oxaloacetate. 2) a-Ketoglutarate accepts the amino group, becoming the amino acid glutamate.

Glutaminase in Acid-Base Balance of Blood

Conversion of Glutamine --> Glutamate + Ammonia Ammonia (base) can act as a buffer to fine tune blood pH.

Aspartate Aminotransferase

Converts Glutamate + Oxaloacetate to α-Ketoglutarate and ASPARTATE

Congenital Hyperammonemia

Deficiency in any urea cycle enzymes (particularly deficiency of OTC) ---> mental retardation, and in severe cases coma and death. Clinical: In blood [Ammonia]↑, [Glutamine] ↑ presence of pyrimidine metabolites (orotic acid) in urine (due to increase in CPSII activity as well as CPSI activity).

Short Term Regulation of Urea Synthesis

The intrahepatic concentration of N-acetylglutamate is high after a protein-rich meal.

Liver and Urea

Liver must pool together all N as Glutamate and Aspartate. Any a.a. that is NOT Asp or Glu must be converted to Asp or Glu. 1) Donates ammonia group from an a.a. to α-ketoglutarate 2) Aminotransferase (requires PLP) makes Glutamate + α-keto acid

Amino Acids that do NOT undergo Transamination

Lys, Thr, & Pro ...are each converted to Glutamate via several steps (NOT a transanimation reaction).

PLP (Vit B6) Deficiency vs Excess

Mild deficiency: Irritability, Nervousness, depression, Hair loss, Insomnia Severe deficiency: Peripheral neuropathy, anemia, Muscle weakness Amount of Vitamin B6 proportional to protein intake Excess vitamin is toxic

Urea Synthesis (in Liver)

NH3 + Asp + HCO3- + 3 ATP --> Urea 3 molecules of ATP but 4 high energy Pi (one from Carbamoyl Phosphate) are used in urea synthesis. Expensive! Urea synthesis uses the "Urea Cycle", involving liver mitochondria and cytosol. Think of this as a base (ammonia) consuming reaction to produce a neutral product (urea). Prevents blood pH from increasing due to ammonia buildup.

NO Action

NO increases conversion of GTP -> cGMP ("c-gimp") cGMP can cause smooth muscle relaxation or can be converted to GMP (used in other reactions)

Result of NO Vasodilation

NO relaxes vascular smooth muscle --> increased blood flow --> male sexual arousal

Source of N & C for Urea Synthesis

Nitrogen from amino acid breakdown is converted to Urea. Urea consists of 2 ammonias (one from Aspartate and one from free ammonia from glutamate) and a CO (from bicarbonate).

Elimination of N by Kidney

No more than 10% N may be eliminated as ammonium ion by kidney. [Remember that excess N circulates in blood mostly as Glutamine (Gln) & Alanine (Ala)] Gln + H2O --> NH3 + Glu Enzyme: Glutaminase Important in acid-base balance.

Vitamin B6

PLP (Pyridoxal Phosphate) essential for all Transamination reactions

Amino Acid Source in Starvation...

Protein breakdown from skeletal muscle is the prominent source of...

Amino Acid Catabolism

You want to get rid of the N so that you can use the Carbons it is attached to for energy and catabolism. Must eliminate nitrogen somehow. Urea is a neutral substance that can store the N until you are ready to eliminate it (urine).

Amino Acid to Urea (basic cycle)

a.a. + α-Ketoglutarate --> Glutamate --> Aspartate + α-Ketoglutarate AND NH4 ---> Urea

CPS I deficiency

autosomal recessive results in Congenital Hyperammonemia Clinical: In blood [Ammonia]↑, [Glutamine] ↑ presence of pyrimidine metabolites (orotic acid) in urine.

Glutamate Dehydrogenase

catalyzes the mitochondrial conversion of Glutamate to Ammonia + α-ketogluarate. (uses H2O and an NAD pr NADP).

When Amino Acid ingested > Body's needs

...Carbons are stored for later use. You must get rid of the excess Nitrogen, leads to higher [urea] in your body due to increased breakdown of amino acid. (eliminate Nitrogen and Store Carbons)

Proline

...is also converted to Glutamate via several steps (NOT a transanimation reaction).

Threonine

...is converted to Ammonia via several steps (NOT a transanimation reaction).

Lysine

...is converted to Glutamate via several steps (NOT a transanimation reaction).

Pre-Urea Cycle Mitochondria

1) Glutamate ---> NH4 and 2) Glutamate + Oxaloacetate ---> Aspartate + α-Ketoglutarate 3) Aspartate leaves the mitochondria to enter Urea Cycle 4) Ammonia (NH4) from Glutamate is transferred to Carbamoyl Phosphate (CPS-1) 5) Carbamoyl Phosphate + Orinithine ---> Citruline 6) Citruline leaves mitochondria to enter Urea Cycle

In Fasting or in Diabetes mellitus

Body is in a state of starvation or perceived starvation (diabetes). Glucose cannot get into the muscles due to lack of insulin. Glucagon is increased bc of low insulin. Overall => catabolic state. Results in state of gluconeogenesis - protein and F.A. breakdown for glucose production. - ingested amino acids primarily used for gluconeogenesis (Acetyl CoA) instead of protein building.

OTC (ornithine transcarbamoylase)

Carbamoyl Phosphate + Orinithine ---> Citruline + Pi (#1 in picture)

Committed step of the Urea Cycle, and is subject to regulation

Carbamoyl Phosphate Synthase (CPS or CPS I)

Glutaminase

Catalyzes: Glutamine + H2O --> NH3 + Glutamate Ammonia released contributes to the 10% ammonia being secreted by the kidneys.

Increase in Glutamine due to Congenital Hyperammonemia

Caused by conversion of Glutamine from Glutamate and Ammonia (see Glutaminase for reverse reaction in normal conditions) in an attempt to eliminate excess ammonia. This can't keep up and ammonia builds up anyways.

Activation of CPS-1

Cellular [Glutamate] is high, signals an excess of free a.a.'s due to protein breakdown or dietary intake. ---> Synthesis of N-acetylglutamate (from Acetyl CoA + Glutamate) ---> Activation of CPS-1

Cirrhosis (definition)

Chronic disease of the liver with gradual destruction of cells and formation of scar tissue; commonly caused by alcoholism

Urea Cycle Overview (Products)

Citrinine and Orthinine cycle through, producing a Urea with each cycle. Uses 1 ATP (Plus 2 ATP and a Pi from Carbamoyl Phosphate). Also produces 1 Fumarate per cycle

Arginase

Cleaves Urea from Arginine, releasing one Urea and Ornithine (which is reused for another urea cycle)

Pyridoxal Phosphate (PL)

Co-factor for aminotransferase enzyme Vitamin B6

Normal dynamic turnover of body proteins

Constant turnover of proteins at all times. 2% of your body's proteins (i.e. muscles) are being broken down to amino acids. Meanwhile 75% of amino acids (from body-protein breakdown and dietary sources) are being reuitilized to build body proteins. 25% of amino acids (diet or body-protein) are catabolized for energy OR potentially building other things (i.e. Acetyl CoA pathways or gluconeogenesis). --> releases Nitrogen (goes to Urea) and Carbons that are converted to Glucose or Acetyl CoA.

NO Synthase

Converts Arginine to Citrulline, releasing NO (Nitrous Oxide) Ca2+ is a Cofactor

Aminotransferase

Converts α-ketoglutarate + Amino Acid --> Glutamate + α-Keto Acid Cofactor is PLP (Pyridoxal Phosphate) Occurs in liver mitochondria. Can also be reversed to make new a.a.'s for protein synthesis.

Viagra (Sildenafil)

Inhibits phosphodiesterase (PDE5) --> prevents conversion of cGMP to GMP and diverts more cGMP to smooth muscle relaxation. This causes increased vasodilation --> increased blood flow --> increased boner.

Hyperammonemia (Adults vs Newborns)

Newborns: due to genetic defect in urea cycle enzyme Adults: due to diseased liver (alcoholism, cancer, hepatitis, etc)

PLP (Vit B6) is Essential for...

- Serotonin synthesis (Trp -> serotonin +CO2) - Glycogen phosphorylase - Heme synthesis - Cys (Cysteine) synthesis - Sphingolipid synthesis - Norepinepherine synthesis (SERa GLYdes HEr Cyster's SPHynx NORth)

Normal Ammonia Processing

In the stomach ("gut"): 1) Intestinal bacteria digest dietary protein, releasing ammonia. 2) Ammonia enters portal vein to liver and is converted to Urea. This causes the ammonia concentration [NH3] in the Portal Blood to be GREATER than that of the Systemic Blood.

Synthesis of N-acetylglutamate

Acetyl CoA + Glutamate Activates CPS-1 INCREASES Urea Production

Major Circulating forms of Amino Acids

Alanine and Glutamine are the major circulating amino acids in blood. Protein breakdown from skeletal muscle is the prominent source of amino acids during starved state.

Major circulating amino acids in blood...

Alanine and Glutamine are the...

N-acetylglutamate

Allosteric activator of Carbamoyl Phosphate Synthase 1. CPS has an ABSOLUTE REQUIREMENT for this activator.

Pre-Urea Cycle Cytosol

Amino Acids + α-ketoglutarate --> Glutamate ---> NH4 and Aspartate + α-Ketoglutarate ------> Urea

Tyrosine Transaminase..

Aminotransferase activity Production of Glutamate from Tyrosine. Catalyzed by.... (uses PLP co-factor)

Urea Cycle (Srini's)

FIGURE 18-10 Urea cycle and reactions that feed amino groups into the cycle. The enzymes catalyzing these reactions (named in the text) are distributed between the mitochondrial matrix and the cytosol. One amino group enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, formed in the matrix by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. The urea cycle consists of four steps. 1 Formation of citrulline from ornithine and carbamoyl phosphate (entry of the first amino group); the citrulline passes into the cytosol. 2 Formation of argininosuccinate through a citrullyl-AMP intermediate (entry of the second amino group). 3 Formation of arginine from argininosuccinate; this reaction releases fumarate, which enters the citric acid cycle. 4 Formation of urea; this reaction also regenerates ornithine. The pathways by which NH4+ arrives in the mitochondrial matrix of hepatocytes were discussed in Section 18.1.

Glutamate vs Gutamine

Glutamine has an EXTRA Amino group. (2 aminos per Glutamine) Glutamine is BASIC Glutamate is ACIDIC

Long Term Regulation of Urea Synthesis

High protein diet --> elevated serum glucagon --> stimulation of the transcription of the genes for urea cycle enzymes

Degradation of Amino Acids

Important under three circumstances: (1) Normal dynamic turnover of body proteins (2) Amino Acid ingested > Body's needs (3) Fasting or in Diabetes mellitus We eliminate all the excess N as urea in the urine. We don't store excess N. Liver is the site of Urea synthesis

Cirrhosis Ammonia Processing

Severe cirrhosis leads to collateral communications between Portal and Systemic veins. Results in increased [NH3] in Systemic Blood ---> leads to Ammonia Toxicity.

Fate of Glutamate produced by aminotransferase...

Some Glutamate is used to produce ammonia for urea (N-disposal) and carbons. The rest is combined with Oxaloacetate to produce Aspartate and α-Ketoglutarate.

Glutamate NOT used for ammonia production...

Some Glutamate is used to produce ammonia. The rest... Glu + Ox.acetate ---> α-Ketoglutarate + Aspartate α-Ketoglutarate - can be reused to produce more Glutamate from other a.a.'s. Aspartate provides the second ammonia in urea. (see prev card)

Treatment of Congenital Hyperammonemia

Specific treatments depend on which urea cycle enzyme is deficient. More general treatments include: 1) limiting protein intake to the amount barely adequate to supply amino acids for growth, while adding to the diet the a-keto acid analogs of essential amino acids. ("a-keto acids" tricks body into thinking there is a lot of glutamate + a-Keto Acid, shifts equilibrium and converts more Glutammate back to a-ketoglutarate + a.a. to reduce ammonia buildup) 2) Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

Transamination

The major mechanism for removing alpha-amino groups from amino acids is via transamination (Fig. 25-3). Transaminases (aminotransferases) catalyze the transfer of an alpha-amino group of an amino acid to the carbon of an alpha-keto acid. The most common alpha-keto acid used as an acceptor of amino groups in these reactions is alphaketoglutarate, with glutamate being the amino acid formed. There are many different aminotransferases, specific for different amino acids, but almost all of which use alphaketoglutarate and L-glutamate as one of the substrate pairs

Source of (free) Ammonia

The only significant reaction in humans to produce ammonia from a.a's is the mitochondrial conversion of Glutamate to Ammonia + α-ketogluarate. (uses H2O and an NAD or NADPH). Enzyme: Glutamate dehydrogenase