The Nitrogen Cycle Nitrite reductase Nitrate reductase nitrogenase

Nitrogen Metabolism Many nitrogen containing compounds eg. Amino acids, nucleotides, porphyrins, neurotransmitters There is no dedicated store for nitrogen or nitrogen compounds in humans

Nonprotein nitrogen derivatives Diet protein Tissue protein Carbohydrate (glucose) transamination Nonprotein nitrogen derivatives Amino nitrogen in glutamate deamination NH3 Urea Acetyl-CoA Citric Acid Cycle CO2 Ketone bodies Amino acid pool Overview of the protein metabolism

OVERVIEW OF AMINO ACID METABOLISM ENVIRONMENT ORGANISM Bio- synthesis Protein Ingested protein 2 3 1 a AMINO ACIDS b c Degradation (required) c Purines Pyrimidines Porphyrins Nitrogen Carbon skeletons Urea (ketogenic) (glucogenic) pyruvate α-ketoglutarate succinyl-CoA fumarate oxaloacetate Used for energy acetoacetate acetyl CoA

Amino acids are the major source of dietary N

NITROGEN BALANCE

Nitrogen intake nitrogen excretion Nitrogen Balance An individual’s nitrogen balance is dependent on a combination of: Nitrogen intake nitrogen excretion Dietary amino acids, nucleotides etc. Urine, faeces, hair and skin loss, perspiration Nitrogen balance status can be: 1) In balance 2) Positive 3) Negative

NITROGEN BALANCE Nitrogen balance = nitrogen ingested - nitrogen excreted (primarily as protein) (primarily as urea) Nitrogen balance = 0 (nitrogen equilibrium) protein ingested = protein excretion Observed in adults Positive nitrogen balance protein ingested > protein excretion during pregnancy, infancy, childhood , body building and recovery from severe illness or surgery Negative nitrogen balance protein ingested < protein excretion starvation, following severe trauma, surgery or infections. Prolonged periods of negative balance are dangerous and fatal if the loss of body protein reaches about one-third of the total body protein

Excess or insufficient dietary amino acid intake leads to the catabolism of amino acids Excess amino acids can be used for energy Insufficient dietary amino acids lead to the catabolism of proteins Insufficient dietary energy leads to the catabolism of proteins For amino acids to be utilised for energy, they must have their a-amino groups removed

Deamination of amino acids Deamination generates: a carbon skeleton a free amino group can be used for anabolic or catabolic reactions generally excreted

Some amino acids can be directly deaminated Serine, threonine and glutamate can be directly deaminated Glutamate deamination is catalysed by glutamate dehydrogenase (GDH)

Glutamine can be deaminated in a two step process Glutamate is then deaminated by GDH glutamine + H2O glutamate + NH3

Glutamine can also be synthesised from glutamate Glutamine synthesis is an energy requiring reaction The reaction is catalysed by glutamine synthetase (GS) glutamate + NH4+ + ATP glutamine + ADP + Pi GS

TRANSAMINATION

Transamination Those amino acids that can not be directly deaminated have their amino groups transferred to specific substrates These substrates are keto acids found in intermediary metabolism a - ketoglutarate oxaloaceatate pyruvate CAC

Addition of amino groups to these keto acids generates amino acids a - ketoglutarate oxaloacetate pyruvate glutamate aspartate alanine Most amino acids are deaminated by donating their a-amino acids to one of these keto acids Thus the deamination of most amino acids leads to the production of either glu, asp, ala or gln.

An example transamination glutamate a-KG a-amino acid a-keto acid glutamate aminotransferase

Pyridoxal phosphate Derived from vitamin B6 Takes part in all amino transferase reactions Forms a Schiff base intermediate with substrates

Role of transamination in metabolism Transamination allows for: 1) the generation of amino acids in short supply 2) the provision of carbon skeletons for energy generation 3) the safe removal of excess amino groups

However when ammonia concentrations are high: Free ammonia is a by-product of brain metabolism glutamate + NH4+ + ATP glutamine + ADP + Pi The neurotransmitter GABA is inactivated by deamination GS GDH a-ketoglutarate + NH4+ + NADPH glutamate + NADP+ + H2O However when ammonia concentrations are high: Brain requires large amounts of ATP This must be generated via oxidative phosphorylation Therefore the CAC must function efficiently GABA – Gamma Amino Butyric Acid

Free ammonia is also produced in muscle Amino groups can be liberated: during normal muscle turnover during starvation during severe muscle activity ATP ADP + Pi 2ADP ATP + AMP AMP IMP + NH4+ AMP deaminase

alanine aminotransferase Pyruvate is usually abundant in active muscle Muscle uses pyruvate as an acceptor keto acid glutamate + pyruvate a-ketoglutarate + alanine alanine aminotransferase Thus in muscle most amino groups are shuttled to alanine (via glutamate) Alanine is then exported to the liver where the amino groups can be liberated

AMP

FATE OF AMINO GROUP DEAMINATION A. Transamination B. Oxidative deamination C. purine nucleotide cycle

A. Transamination Transamination by Aminotransferase (transaminase) always involve PLP coenzyme (pyridoxal phosphate) reaction goes via a Schiff’s base intermediate all transaminase reactions are reversible

Aminotransferases Aminotransferases can have specificity for the alpha-keto acid or the amino acid Aminotransferases exist for all amino acids except proline and lysine The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and a-ketoglutarate, which participate in reactions with many different aminotransferases to an alpha-keto acid  alpha-amino acid

Transamination aminotransferases

Glu+pyruvate glutamate-pyruvate aminotransferase GPT, ALT -Ketoglutarate+Ala Glu+Oxaloacetate Glutamic oxaloacetictransaminase GOT, AST -Ketoglutarate+Asp *** ALT and AST are components of a "liver function test". Levels increase with damage to liver (cirrhosis, hepatitis) or muscle (trauma)

The mechanism of transamination + PLP AA –H2O +H2O Schiff’s base

Schiff’s base isomer Molecule rearrange PMP –H2O +H2O -ketoacid +

Transamination Interconversion of amino acids Collection of N as glu Provision of C-skeletons for catabolism

B. Oxidative Deamination L-glutamate dehydrogenase (in mitochondria) Glu + NAD+ (or NADP+) + H2O  NH4+ + a-ketoglutarate + NAD(P)H +H+ Requires NAD+ or NADP + as a cofactor Plays a central role in AA metabolism ?

It is inhibited by GTP and ATP, and activated by GDP and ADP urea cycle ? It is inhibited by GTP and ATP, and activated by GDP and ADP

Combined Deamination ?

Transamination + Oxidative Deamination Combined deamination ＝ Transamination + Oxidative Deamination The major pathway ！！！

C. purine nucleotide cycle AMP NH3 AA Asp IMP -Keto glutarate H2O aminotransferases AST C. purine nucleotide cycle AMP -Keto acid Oxaloacetate fumarate malate

The metabolism of α-ketoacid Biosynthesis of nonessential amino acids TCA cycle member + amino acid α-keto acid + nonessential amino acid A source of energy (10%) ( CO2+H2O ) Glucogenesis and ketogenesis

SUMMARY Nitrogen balance status depends on the intake and use of N containing compounds Excess N from amino acids must be excreted A series of aminotransferase and deamination reactions shuttle nitrogen to appropriate molecules and tissues Brain and muscle can generate large amounts of excess nitrogen as part of their metabolism The liver is an important tissue for processing excess nitrogen