Amino Acid Catabolism: N

Overview of Carbon Flow:

Carbons from the pink amino acids can all participate in gluconeogenesis Carbons from the blue amino acids can all end up as acetoacetyl-CoA and, if in excess, can produce ketone bodies Trp, Phe, Tyr, Ile are in both categories Only Leu and Lys are solely ketogenic

Glucogenic and Ketogenic Amino Acids

Glucogenic vs Ketogenic

Amino acids degraded to pyruvate, ketoglutarate, succinyl~CoA, fumarate, or oxaloacetate are called glucogenic because these intermediates can be used in gluconeogenesis

Amino acids degraded to acetyl~CoA or acetoacetate are called ketogenic. Many fit both categories; however, leucine and lysine are exclusively ketogenic.

Nitrogen and Carbon Go Separate Ways

H R1 C COO NH3 Transaminase H R1 C COO O

+ -

+ O

R2

C COO

Transaminases (aminotransferases) catalyze the reversible reaction at right.

+ R2 NH3
+

C COO

There are multiple transaminase enzymes which vary in substrate specificity.

Some show preference for particular amino acids or classes of amino acids as amino group donors, and/or for particular -keto acid acceptors.

COO CH2 CH2 CH2 O C O CH2 COO CH2

COO

COO

CH2

HC

NH3+

+
C

HC

NH3+

COO

COO

COO

COO

aspartate -ketoglutarate oxaloacetate glutamate

Aminotransferase (Transaminase)

Example of a Transaminase reaction:

Aspartate donates its amino group, becoming the -keto acid oxaloacetate.

-Ketoglutarate accepts the amino group, becoming the amino acid glutamate.

COO CH2 CH2 CH3 O C O CH2 CH2

COO

CH3

HC

NH3+

+
C

HC

NH3+ COO

COO

COO

COO

alanine

-ketoglutarate

pyruvate

glutamate

Aminotransferase (Transaminase)

In another example, alanine becomes pyruvate as the amino group is transferred to -ketoglutarate.

Transaminases equilibrate amino groups among available -keto acids.

This permits synthesis of non-essential amino acids, using amino groups from other amino acids & carbon skeletons synthesized in a cell.

Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized.

Although the amino N of one amino acid can be used to synthesize another amino acid, N must be obtained in the diet as amino acids (proteins).

Essential amino acids must be consumed in the diet.

Mammalian cells lack enzymes to synthesize their carbon skeletons (-keto acids). These include:

Isoleucine, leucine, & valine

Lysine

Threonine

Tryptophan

Phenylalanine (Tyr can be made from Phe.)

Methionine (Cys can be made from Met.)

Histidine (Essential for infants.)

H O P O + N H O CH3 H2 C OH O C

pyridoxal phosphate (PLP)

The prosthetic group of Transaminase is pyridoxal phosphate (PLP), a derivative of vitamin B6.

R NH2 N+ O HC H2 C O H P O + N H O CH3 O (CH2)4

H C COO Enz

Amino acid

Enzyme (Lys)-PLP Schiff base

In the resting state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the -amino group of an enzyme lysine side-chain.

EnzLysNH2 R N+ O P O O + N H CH3 H2 C O HC H O H C COO

The -amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP.

Amino acid-PLP Shiff base (aldimine)

The active site lysine extracts H+, promoting tautomerization, followed by reprotonation & hydrolysis.

Transferring amino groups requires pyridoxal phosphate (PLP), derived from vitamin B6

AAT with PLP in aldimine linkage to Lys258

O EnzLysNH2 NH2 CH2 H2 C OH R O P O O + N CH3 H Pyridoxamine phosphate (PMP) O C COO -keto acid

What was an amino acid leaves as an -keto acid.

The amino group remains on what is now pyridoxamine phosphate (PMP). A different -keto acid reacts with PMP and the process reverses, to complete the reaction.

Transamination is a Ping-Pong Reaction

The enzymes that catalyze it are called both transaminases and aminotransferases In a ping-pong reaction, the first product must leave the active site before the second substrate can bind In this case, the amino group is transiently (but covalently) held by the prosthetic group pyridoxal phosphate (as pyridoxamine phosphate)

Aspartate Aminotransferase (AST)

PLP (red) is in the active site of one of the two subunits of this dimeric transaminase They are named after the amino group donor

Amino-group Transfers Using Pyridoxal Phosphate (from Vitamin B6) Play a Key Role

EnzLysNH2 R N+ O HC H O H2 C P O + N H CH3 O H C COO

Amino acid-PLP Shiff base (aldimine)

Several other enzymes that catalyze metabolism or synthesis of amino acids also utilize PLP as prosthetic group, and have mechanisms involving a Schiff base linkage of the amino group to PLP.

In addition to equilibrating amino groups among available -keto acids, transaminases funnel amino groups from excess dietary amino acids to those amino acids (e.g., glutamate) that can be deaminated.

Carbon skeletons of deaminated amino acids can be catabolized for energy, or used to synthesize glucose or fatty acids for energy storage.

Only a few amino acids are deaminated directly.

Glutamate and Glutamine are Important Nitrogen Carriers

Most amino acid metabolism occurs in the liver, where In the cytosol, -ketoglutarate receives amino groups transferred from other amino acids, to form glutamate(transamination) Glutamate and glutamine then pass into liver mitochondria for further metabolism Glutamine is the common ammonia carrier from other tissues by transamination of glutamate (except muscle, where alanine is used why?)

How Ammonia from Tissues is Transported by Glutamine in the Bloodstream

The enzyme glutamine synthetase, which plays a central metabolic role in all organisms, can transfer free ammonia to glutamate, thus producing glutamine This two-step reaction (another one!) requires an activated phosphorylated intermediate Glutamine thus carries two amino groups, is nontoxic and highly soluble, and is present in blood at higher levels than other amino acids Once in mitochondria, glutaminase regenerates glutamate and ammonia

NH3+ COO NAD(P)+ NAD(P)H COO + NH4+

H2 H2 OOC C C C glutamate H
H2O

O H2 H2 OOC C C C -ketoglutarate

Glutamate Dehydrogenase catalyzes a major reaction that effects net removal of N from the amino acid pool.

Glutamate Dehydrogenase

It is one of the few enzymes that can use NAD+ or NADP+ as e acceptor.

Oxidation at the -carbon is followed by hydrolysis, releasing NH4+.

Oxidative Deamination in Liver Mitochondria

As the recipient of amino groups from many sources, glutamate now sheds it as ammonia for excretion, and the product -ketoglutarate can recycle as a nitrogen acceptor, enter the TCA cycle, or serve as a precursor in gluconeogenesis

Amino acid

-ketoglutarate NADH + NH4

+

-keto acid glutamate

+

NAD + H2O

Transaminase

Glutamate Dehydrogenase

Summarized above:

The role of transaminases in funneling amino N to glutamate, which is deaminated via Glutamate Dehydrogenase, producing NH4+.

Another Way to Get Ammonia to the Liver: Glucose-Alanine Cycle

Anerobic muscle contraction yields pyruvate (from glycolysis) and amino groups (from protein breakdown) Alanine aminotransferase makes alanine, which goes to the liver where it is reconverted to pyruvate (and then to glucose, via gluconeogenesis, to be exported back to muscle) This complements the Cori cycle with lactate And ammonia is excreted from the liver

Why does this make metabolic sense for liver and muscle function?

H2O O C COO H2C NH3+ C COO H3C

+ H2O NH4

HO

CH2

H C

COO

NH3+

serine

aminoacrylate

pyruvate

Serine Dehydratase

Some other pathways for deamination of amino acids:

1. Serine Dehydratase catalyzes: serine pyruvate + NH4+ 2. Peroxisomal L- and D-amino acid oxidases catalyze: amino acid + FAD + H2O -keto acid + NH4+ + FADH2 FADH2 + O2 FAD + H2O2 2 H2O + O2

Catalase catalyzes: 2 H2O2

How to Get Rid of Toxic Ammonia?

Glutamine
(muscle and other tissues) (muscle)

Amino acids

Alanine

(ingested protein)

Only urea output is proportional to protein in the diet

Urea Uric acid

Fig. 18-2b

Excrete it, as:

Creatinine

Ammonium ion

Mmol-N/day 40 (gm/day) (0.7)

800 (25)

15 (0.8)

40 (1.5)

O H2N C NH2

urea

Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it.

Urea is less toxic than ammonia.

The Urea Cycle occurs mainly in liver.

The 2 nitrogen atoms of urea enter the Urea Cycle as NH3 (produced mainly via Glutamate Dehydrogenase) and as the amino N of aspartate.

The NH3 and HCO3 (carbonyl C) that will be part of urea are incorporated first into carbamoyl phosphate.

Whats So Great About Urea?

Urea synthesis is essential to humans, because

We require protein in our diet to provide amino acids Which cant be stored, unlike fats and carbohydrates The resultant excess nitrogen after conversion to ammonia must be excreted as urea, or It will circulate in the bloodstream and be toxic if it rises above certain levels

Urea is compact (and symmetric),

2 of its 8 atoms are nitrogen (what % by weight?) it is very water soluble while being nontoxic

Urea is not so great for birds and terrestrial reptiles

excretion requires large water loss, so these uricoteles eliminate crystalline uric acid

Amino Acids vs. Urea vs. Ammonia

micromolar

Normal plasma levels in N-equivalents

103 554 436 9,200 22 (10-50)

L-Glutamic Acid L-Glutamine L-Alanine Urea NH3

These values demonstrate the efficiency of the conversion of ammonia to urea. Luckily for us!

Once Safely in the Liver, How is Ammonia Converted to Urea for Excretion?

HCO3 ATP ADP O HO NH3 Pi O H2N ATP ADP O H2N C OPO32 C O C OPO32

Carbamoyl Phosphate Synthase (Type I) catalyzes a 3-step reaction, with carbonyl phosphate and carbamate intermediates.

carbonyl phosphate

Ammonia is the N input.

The reaction, which involves cleavage of 2 ~P bonds of ATP, is essentially irreversible.

carbamate

carbamoyl phosphate

HCO3 ATP ADP O HO NH3 Pi O H2N ATP ADP O H2N C OPO32 C O C OPO32

Alternate forms of Carbamoyl Phosphate Synthase (Types II & III) initially generate ammonia by hydrolysis of glutamine.

carbonyl phosphate

The type II enzyme includes a long internal tunnel through which ammonia & reaction intermediates such as carbamate pass from one active site to another.

carbamate

carbamoyl phosphate

HCO3 + NH3 + 2 ATP

O C OPO32 + 2 ADP + Pi

Carbamoyl Phosphate Synthase

H2N

carbamoyl phosphate

Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation.

glutamate (Glu)
O H N H CH2 CH2 COO C COO

N-acetylglutamate
H3C C

H COO

H3N+

CH2

CH2

COO

Carbamoyl Phosphate Synthase has an absolute requirement for an allosteric activator N-acetylglutamate.

This derivative of glutamate is synthesized from acetyl-CoA & glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.

O O OPO32 NH CH2 Pi CH2 HC COO ATP AMP + PPi NH3+ COO CH2 CH2 C NH2 NH3+ H2N C CH2 CH2 CH2 HC COO

Urea Cycle
carbamoyl phosphate citrulline 1
NH3+

Urea Cycle
2
COO

Enzymes in mitochondria: 1. Ornithine Transcarbamylase

ornithine
O H2N C NH2 H2N C NH CH2 COO CH2 CH2 HC NH3+ COO HC CH COO NH2+ HC

NH2 COO

4
H2O

aspartate
CH2

urea

HC

H N COO

C NH CH2 CH2 CH2 HC

NH2+

arginine

argininosuccinate
NH3+

Enzymes in cytosol: 2. ArgininoSuccinate Synthase 3. Argininosuccinase 4. Arginase.

fumarate

COO

cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline ornithine arginine fumarate citrulline aspartate argininosuccinate

urea

For each cycle, citrulline must leave the mitochondria, and ornithine must enter the mitochondrial matrix. An ornithine/citrulline transporter in the inner mitochondrial membrane facilitates transmembrane fluxes of citrulline & ornithine.

cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline ornithine arginine fumarate citrulline aspartate argininosuccinate

urea

A complete Krebs Cycle functions only within mitochondria. But cytosolic isozymes of some Krebs Cycle enzymes are involved in regenerating aspartate from fumarate.

COO CH2 CH2 CH2 O C O CH2 COO CH2

COO

COO

CH2

HC

NH3+

+
C

+
COO

HC

NH3+

COO

COO

COO

aspartate -ketoglutarate oxaloacetate glutamate

Aminotransferase (Transaminase)

Fumarate is converted to oxaloacetate via Krebs Cycle enzymes Fumarase & Malate Dehydrogenase. Oxaloacetate is converted to aspartate via transamination (e.g., from glutamate). Aspartate then reenters Urea Cycle, carrying an amino group derived from another amino acid.

How is Ammonia Converted to Urea for Excretion?

And it costs 3 ATPs

Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood.

Total lack of any Urea Cycle enzyme is lethal.

Elevated ammonia is toxic, especially to the brain.

If not treated immediately after birth, severe mental retardation results.

Postulated mechanisms for toxicity of high [ammonia]:

1. High [NH3] would drive Glutamine Synthase: glutamate + ATP + NH3 glutamine + ADP + Pi

This would deplete glutamate a neurotransmitter & precursor for synthesis of the neurotransmitter GABA.

2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P)+ -ketoglutarate + NAD(P)H + NH4+ The resulting depletion of -ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

Treatment of deficiency of Urea Cycle enzymes (depends on which enzyme is deficient):

limiting protein intake to the amount barely adequate to supply amino acids for growth, while adding to the diet the -keto acid analogs of essential amino acids.

Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline

The complete Urea Cycle is significantly only in liver.

ornithine citrulline However some urea aspartate enzymes of the arginine argininosuccinate pathway are in fumarate other cells and tissues where they generate arginine & ornithine, which are precursors for other important molecules. E.g., Argininosuccinate Synthase, which catalyzes synthesis of the precursor to arginine, is in most tissues. Mitochondrial Arginase II, distinct from the cytosolic Urea Cycle Arginase, cleaves arginine to yield ornithine.

NH2 NH2 C NADP+ CH2 H 2O CH2

+

NH2 N 1/2 NADPH 1/2 NADP+ CH2 O2 H2O CH2 CH2 COO
+

C OH NH + NO NH

NH2

NH

NADPH

CH2 CH2

CH2

O2

CH2 H3N CH H 3N

H3N

CH

COO

CH

COO

arginine Nitric Oxide Synthase

hydroxyarginine

citrulline

Arginine is a constituent of proteins, & is precursor for synthesis of creatine and nitric oxide (NO). NO is a short-lived signal molecule with diverse roles in different cell types: regulating smooth muscle contraction, gene transcription, metabolism, & neurotransmission. Many effects of NO arise from its activation of a soluble cytosolic Guanylate Cyclase that synthesizes cyclic-GMP.

H3N CH2

CH2

CH2

CH2

NH3+

putrescine
CH2 CH2 CH2 NH3+

H3N

CH2

CH2

CH2 NH

CH2

spermidine

Polyamines include putrescine, spermidine, spermine.

Ornithine is a major precursor for synthesis of polyamines.

Conversion of ornithine to putrescine is catalyzed by Ornithine Decarboxylase.

The cationic polyamines have diverse roles in cell growth & proliferation.

Disruption of polyamine synthesis or metabolism leads to disease in animals & humans.