Amino Acid Metabolism - Nitrogen Economy and Urea Cycle

Includes information from all slides; does not include main urea cycle pathway

Dietary proteins are digested to absorbable forms, known as…

amino acids, dipeptides, and tripeptides

What is the exception to protein digestion?

Antibodies in maternal milk are taken up intact by infants

Where does digestion of dietary proteins begin?

In the stomach. Proteins are the only macromolecule whose digestion begins in the stomach.

What are the main enzymes involved in digestion of proteins in the stomach?

Pepsin and rennin

Pepsin is found in adult stomach, while rennin is present in infants

Pepsin - secretion, optimum pH, products, type of enzyme

Pepsin, an endonuclease, is secreted by chief cells in the inactive form - pepsinogen - which is activated by HCl (which is secreted by parietal cells). Pepsinogen can also be activated auto-catalytically by another pepsin molecule. pH optimum is 2.0 to 3.0. It produces peptides and a few amino acids.

Rennin - secretion, optimum pH, products, type of enzyme

Rennin is present in infants and secreted as pro-rennin. pH optimum is 4.0. Acts on casein of milk, which is involved in the curdling of milk.

Hyperchlorhydria

Increased HCl levels due to increased gastrin production

Achlorhydria/hypochlorhydria

Decreased HCl levels, due to pernicious anemia - autoimmune destruction of parietal cells; antacids; gastric bypass procedures, etc)

Digestion in the small intestine (duodenum) is done by which enzymes

pancreatic proteases (specific for different side chain groups of AA), endopeptidases, and exopeptidases

What are the endopeptidases involved in digestion of proteins in the small intestine?

Trypsin, chymotrypsin, elastase, and collagenase

What are the exopeptidases involved in digestion of proteins in the small intestine?

Carboxypolypeptidases A and B

What are the pro-enzymes/zymogens of the enzymes involved in digestion in the small intestine?

Trypsinogen → trypsin (activated by intestinal brush-border enzyme enterokinase)

chymotrypsinogen → chymotrypsin

Procarboxypeptidases A and B → carboxypeptidases A and B

Proelastase → elastase

Rest of the zymogens are activated by trypsin

Release and activation of proteases from small intestine is mediated by

cholecystokinin and secretin - GI hormones

What are the peptidases in the enterocytes that line the small intestinal villi?

Aminopolypeptidases and dipeptidases

Both are exopeptidases

Hydrolyze one amino acid at a time from the N-terminal ends

They produce tripeptides, dipeptides, and amino acids

How are L-amino acids absorbed into the cell?

Via secondary active transport using Na+-AA cotransporters

There are 4 separate cotransporters for neutral, acidic, basic, and glycine/imino amino acids.

Once in the cell, the cross the basolateral membrane into the blood via facilitative diffusion

How are dipeptides and tripeptides absorbed into the cell?

Via secondary active transport using H+-dependent cotransporters

Inside the cell, the peptides are hydrolyzed to amino acids by cytosolic peptidases

They cross the basolateral membrane into the blood by facilitative diffusion 

Hartnup disease - cause

Defect in the absorption of non polar amino acids

AR mutation in SLC6A19 gene

Hartnup disease - pathogenesis

Defective absorption of neutral AA (I, L, F, T, W, V, A, S, Y) from epithelial cells of intestines and kidneys

Deficient W → deficient serotonin, melatonin, and niacin

Hartnup disease - symptoms

pellagra-like symptoms (diarrhea, dementia (and other neurological symptoms), dermatitis), aminoaciduria

Infant form: photosensitivity, intermittent ataxia, tremor

Cystinuria - cause

Defect in the transport of cysteine, ornithine, lysine, and arginine (COLA)

AR mutation in SLC3A1 and SLC7A9

Cystinuria - pathogenesis

defective transport in kidney and intestine

cysteine molecules are not filtered by the kidney can be oxidized in urine to form cystine → form hexagonal white crystals, which grow into pink or yellow stones

Cystinuria - symptoms

kidney stones - may block urinary tract, aminoaciduria (but no symptoms of AA deficiency)

Summary of digestion and absorption of amino acids

Nitrogen balance

What is the amino acid pool? Explain the input and output of amino acids.

Free amino acids are present throughout the body (normally input = output)

Supplied by (input):

• Exogenous (dietary) protein

• Nonessential AA synthesized from simple intermediates

• Degradation of body proteins

Used for (output): 

• Synthesis of body protein

• Synthesis of nitrogen-containing small molecules

• Synthesis of glucose, fatty acids, and ketones

• Oxidation to CO2 and H2O

What cofactor is required for active transport into tissues?

Amino acids are transported into tissues actively using PLP (vitamin B6)

Protein turnover

Most proteins are constantly being synthesized and degraded (~300-400 g/day)

Catabolism of amino acids is important for

synthesis of many other molecules and energy

What modifications must be done to amino acids before they can be used for other pathways or energy?

AA must first undergo removal of nitrogen. The a-NH3 group prevents oxidation of AA.

Nitrogen can be incorporated into other compounds or excreted (as urea)

How are carbon skeletons of amino acids metabolized?

Various pathways:

• Glucogenic AA form pyruvate or intermediates of TCA cycle and can enter gluconeogenesis or energy synthesis

• Ketogenic AA form acetyl-CoA, acetoacetyl CoA, or acetoacetate and can enter ketogenesis or energy synthesis

• Binding of amino acids to carbon skeletons forms new AA

Removal of nitrogen from amino acids involves two steps:

Transamination and deamination

1. Transamination: transfer of a-amino group to a-ketoacid producing a new a-amino acid and new a-ketoacid

2. Deamination: oxidative deamination (requires molecular oxygen) and non-oxidative deamidation

Describe the transamination reaction of the removal of nitrogen from amino acids

• reversible reaction

• a-NH2 group of one a-amino acid (donor AA) is transferred to a a-ketoacid (acceptor) resulting in formation of a new a-amino acid and a new a-ketoacid

  • Exists for all amino acids except lysine and threonine

In transamination reaction, the acceptor is a ___ and the new AA is ___. Reaction is catalyzed by ___. 

The acceptor is a-ketoglutarate and the new AA is glutamate. Reaction is catalyzed by transaminases (aka aminotransferases)

Where are aminotransferases located? What is the required coenzyme for this reaction?

Present in almost all mammalian tissues, in both cytosol and mitochondria. It is named after amino acid donor. Aminotransferases require PLP (B6) and there is no free ammonia formation from this reaction. 

What are the two transaminases of clinical importance?

Alanine transaminase (ALT, glutamate-pyruvate transaminase) and aspartate transaminase (AST, glutamate-oxaloacetate transaminase)

What does elevated ALT or AST mean?

Small amounts detected in blood reflects damage to tissues rich in these enzymes (such as liver)

AST is also used as cardiac marker

Describe the oxidative deamination reaction of the removal of nitrogen from amino acids (GDH)

The amino groups of most amino acids are ultimately funneled to glutamate → the only AA that undergoes rapid oxidative deamination

Enzyme: glutamate dehydrogenase (GDH) or L-amino acid oxidase and D-amino acid oxidase

Glutamate dehydrogenase reaction

Reversible reaction that occurs in the mitochondria of liver and kidneys

Glutamate → a-ketogluterate

• Requires NAD+

• Stimulated by ADP, GDP

A-ketogluterate → glutamate

• Requires NADPH

• Stimulated by ATP, GTP

Releases free ammonia that enters the urea cycle

Describe the oxidative deamination reaction of the removal of nitrogen from amino acids (L/D-AA oxidase)

Enzyme: L-amino acid oxidase (requires FMN) and D-amino acid oxidase (requires FAD)

Present in peroxisomes of liver and kidneys

Produces H2O2, NH4, and keto acids

Describe the non-oxidative deamination reaction of the removal of nitrogen from amino acids. (S, hS, T)

Hydroxy amino acids deamination (AA with one or more hydroxyl groups)

Includes: serine, homoserine, threonine

Enzymes - ~amino acid~ dehydratases

• Requires PLP as cofactor

• Releases NH4

Describe the non-oxidative deamination reaction of the removal of nitrogen from amino acids. (N, Q)

Hydrolytic deamination (AA with amide groups)

Includes: asparagine and glutamine

Enzymes: hydrolases: 

• Glutaminase: converts Q → E + NH3 (in the liver, enters urea cycle)

• Asparaginase: converts N → D + NH3

Describe the release of ammonia in most tissues

AA transamination produces glutamate → glutamine (tranport form of ammonia) → to the kidneys (buffering), liver (urea formation), intestine (used as fuel)

Describe the release of ammonia in muscle

AA transamination produces alanine (transport form of ammonia to liver) → produces glutamine (as other tissues) → can utilize branched chain amino acids

Describe the release of ammonia in intestines

Predominantly use glutamine and asparagine for energy → release ammonia directly to portal system

What are the major carriers of nitrogen in the blood?

Alanine and glutamine

What are the misc. sources of ammonia for the urea cycle?

• dehydratase reactions of serine and threonine

• deamination of histidine

• purine nucleotide degradation in muscle and brain

• intestinal bacteria

What is urea?

The major form of excreted nitrogen in humans - water-soluble and nontoxic. 

Where is urea produced?

In the liver, both mitochondria and cytosol, by the urea cycle

What are the donors of ammonia for urea?

1 nitrogen is from free NH3 (released from glutamate by glutamate dehydrogenase)

2 nitrogen are from aspartate (formed from transamination of glutamate by AST)

Glutamate is the final carrier of NH3

The C and O of urea are from CO2 (as HCO3-)

Symptoms of hyperammonemia (in general)

Tremors, slurred speech, somnolence, vomiting, cerebral edema, blurred vision, usually associated with decreased blood urea levels

Causes of hyperammonemia

Acquired: liver disease (acute, chronic)

Hereditary: genetic defects of urea cycle

Relationship between hyperammonemia and BUN levels

Usually, hyperammonemia is associated with decreased BUN levels

In renal failure → increased blood urea b/c it is unable to exert → diffuses into gut → urease → increased ammonia → secondary hyperammonemia

What can be used to treat/limit secondary hyperammonemia as a result of renal failure?

Oral neomycin

In renal failure → increased blood urea b/c it is unable to exert → diffuses into gut → urease → increased ammonia → secondary hyperammonemia

Causes of ammonia toxicity (3 possible results)

Increased NH3 concentration:

1. Enhances utilization of a-KG depressing TCA cycle

2. Enhances glutamine formation from glutamate

   1. Decreased formation of GABA

   2. Cerebral edema d/t glutamine-induced osmotic shifts

   3. Increased outflow of glutamine from brain cells → entry of tryptophan into brain cells → increased serotonin

3. NH3 is basic → alkalization of intracellular compartment

Ornithine transcarbamoylase deficiency - causes

X-linked recessive defect in OTC

Ornithine transcarbamoylase deficiency - symptoms

Usually observed first few days of life

• cerebral edema, lethargy, convulsions, coma

Ornithine transcarbamoylase deficiency - lab presentation

• Increased NH3

• Decreased BUN

• increased blood glutamine

• Orotic acuduria: increased blood orotic acid level because carbamoyl phosphate is accumulated → travels to cytosol → enters process of synthesis of pyrimidine nucleotides → intermediate orotic acid is accumulated

Carbamoyl phosphate synthetase deficiency - cause

Autosomal recessive defect in CPSI

Carbamoyl phosphate synthetase deficiency - symptoms

Same as ornithine transcarbamoylase deficiency

Usually observed first few days of life

• cerebral edema, lethargy, convulsions, coma

Carbamoyl phosphate synthetase deficiency - lab presentation

• Increased NH3

• Decreased BUN

• Increased blood glutamine

• NO OROTIC ACIDURIA

Urea cycle enzyme deficiencies - citrullinemia

Defect in arginosuccinate synthase

Citrullinuria

Urea cycle enzyme deficiencies - arginosuccinic aciduria

Defect in arginosuccinate lyase

Arginosuccinic acid (arginosuccinate) is found in blood, CSF, and urine

Urea cycle enzyme deficiencies - hyperargininemia

Defect in arginase

Tx: diet without R

Treatment of hyperammonemia may include

• Decreased protein intake

• Removal of ammonia

• Sodium benzoate + glycine → hippuric acid → excretion

  • Phenylacetate + glutamine → phenylacetyl glutamine → excretionS

Summary of amino acid metabolism

Summary of urea cycle