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Amino Acid Catabolism: N

Copyright 1999-2008 by Joyce J. Diwan.

All rights reserved.

Molecular Biochemistry II

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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.

H

R1 C COO-

+ R2 C COO-

NH3+

O

Transaminase

H

R1 C COO

-

+ R2 C COO

-

O NH3+

Transaminases(aminotransferases)

catalyze the

reversible reaction

at right.

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Example of a Transaminase reaction:

Aspartate donates its amino group, becoming the

a-keto acid oxaloacetate.

a-Ketoglutarate accepts the amino group,

becoming the amino acid glutamate.

aspartate a-ketoglutarate oxaloacetate glutamate

Aminotransferase (Transaminase)

COO

CH2

CH2

C

COO

O

COO

CH2

HC

COO

NH3+

COO

CH2

CH2

HC

COO

NH3+

COO

CH2

C

COO

O+ +

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In another example, alanine becomes pyruvate as

the amino group is transferred to a-ketoglutarate.

alanine a-ketoglutarate pyruvate glutamate

Aminotransferase (Transaminase)

COO

CH2

CH2

C

COO

O

CH3

HC

COO

NH3+

COO

CH2

CH2

HC

COO

NH3+

CH3

C

COO

O+ +

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Transaminases equilibrate amino groups among

available a-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).

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Essential amino acids must be consumed in the diet.

Mammalian cells lack enzymes to synthesize theircarbon skeletons (a-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.)

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The prosthetic group of Transaminase is

pyridoxal phosphate (PLP), a derivative of

vitamin B6.

pyridoxal phosphate (PLP)

NH

CO

P

OO

O

OH

CH3

CH O

H2

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In the resting state, the aldehyde group of pyridoxal

phosphate is in a Schiff base linkage to the e-amino

group of an enzymelysine

side-chain.

NH

C

O

P

OO

O

O

CH3

HC

H2

N

(CH2)4

Enz

H

+

RHC COO

NH2

Enzyme (Lys)-PLP Schiff base

Amino acid

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The active site lysine extracts H+, promoting

tautomerization, followed by reprotonation & hydrolysis.

NH

CO

P

OO

O

O

CH3

HC

H2

N

HC

H

+

R COOEnzLysNH2

Amino acid-PLP Shiff base (aldimine)

The a-amino group

of a substrate amino

aciddisplaces the

enzyme lysine, toform a Schiff base

linkage to PLP.

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The amino group remains on what is now pyridoxamine

phosphate (PMP).

A different a-keto acid reacts with PMP and the process

reverses, to complete the reaction.

NH

CO

P

OO

O

OH

CH3

CH2

NH2

H2

R C COO

O

EnzLysNH2

Pyridoxamine phosphate (PMP)

a-keto acid

What was anamino acid

leaves as an

-keto acid.

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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.

NH

CO

P

OO

O

O

CH3

HC

H2

N

HC

H

+

R COOEnzLysNH2

Amino acid-PLP Shiff base (aldimine)

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Chime Exercise

Two neighboring students or student groups should

team up, each displaying one of the following:

Transaminase with PLP in Schiff base linkage to

the active site lysine residue.

Transaminase in the PMP form, with glutarate, an

analog ofa-ketoglutarate, at the active site.

Students should then show and explain the structure

displayed by them to the neighboring student or

student group.

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In addition to equilibrating amino groups among

available a-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.

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

Oxidation at the a-carbon is followed by hydrolysis,

releasing NH4+.

OOC

H2C

H2C C COO

O

+ NH4+

NAD(P)+

NAD(P)H

OOC

H2C

H2C C COO

NH3+

Hglutamate

a-ketoglutarate

Glutamate Dehydrogenase

H2OGlutamateDehydrogenase

catalyzes a major

reaction that effects

net removal of Nfrom the amino

acid pool.

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Summarized above:The role of transaminases in funneling amino N to

glutamate, which is deaminated via Glutamate

Dehydrogenase, producing NH4+.

Amino acid -ketoglutarate NADH + NH4+

-keto acid glutamate NAD+

+ H2O

Transaminase Glutamate Dehydrogenase

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Some other pathways for deamination of amino acids:

1. Serine Dehydratase catalyzes:

serine pyruvate + NH4+

2. PeroxisomalL- and D-amino acid oxidases catalyze:

amino acid + FAD + H2O -keto acid + NH4

+ + FADH2

FADH2 + O2 FAD + H2O2

Catalase catalyzes: 2

H2O2 2 H2O + O2

HO CH2HC COO

NH3

+

C COO

OH2O NH4+

C COO

NH3

+

H2C H3C

H2O

serine aminoacrylate pyruvate

Serine Dehydratase

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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.

H2N C

O

NH2

urea

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Carbamoyl Phosphate

Synthase (Type I) catalyzes

a 3-step reaction, with

carbonyl phosphate and

carbamate intermediates.

Ammonia is the N input.

The reaction, which

involves cleavage of2 ~Pbonds of ATP, is essentially

irreversible.H2N C OPO3

2

O

H2N C O

O

HO C

O

OPO32

HCO3

ATP

NH3

ADP

ATP

Pi

ADP

carbonyl phosphate

carbamate

carbamoyl phosphate

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Alternate forms of

Carbamoyl Phosphate

Synthase (Types II & III)

initially generate ammonia

by hydrolysis ofglutamine.

The type II enzyme includes

a long internal tunnel

through which ammonia &

reaction intermediates such

as carbamate pass from one

active site to another. H2N C OPO32

O

H2N C O

O

HO C

O

OPO32

HCO3

ATP

NH3

ADP

ATP

Pi

ADP

carbonyl phosphate

carbamate

carbamoyl phosphate

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Carbamoyl Phosphate Synthase is the committed step

of the Urea Cycle, and is subject to regulation.

H2N C OPO32

O

HCO3 + NH3 + 2ATP

+ 2ADP + Pi

Carbamoyl PhosphateSynthase

carbamoyl phosphate

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Carbamoyl Phosphate Synthase has an absolute

requirement for an allostericactivatorN-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.

H3N+

C COO

CH2

CH2

COO

H

glutamate (Glu)

NH

C COO

CH2

CH2

COO

H

CH3C

O

N-acetylglutamate

O CO NH

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H2N C OPO32

O

CH2

CH2

CH2

HC

COO

NH3+

NH3+

CH2

CH2

CH2

HC

COO

NH3+

NH

CO NH2

COO

CH2

HC

COO

NH2

CH2

CH2

CH2

HC

COO

NH3+

NH

C NH2+

COO

CH2

HC

COO

HN

AMP + PPi

ATP

CH2

CH2

CH2

HC

COO

NH3+

NH

C

NH2+H2N

COO

HC

CH

COO

C NH2H2N

O H2O

Pi

ornithine

urea

citrulline

aspartate

arginino-succinate

fumarate

arginine

carbamoylphosphate

Urea Cycle

1

2

3

4

Urea Cycle

Enzymes in

mitochondria:1. Ornithine

Trans-

carbamylase

Enzymes in

cytosol:

2. Arginino-

Succinate

Synthase

3. Arginino-

succinase

4. Arginase.

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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 citrulline

urea aspartate

arginine argininosuccinate

fumarate

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A complete Krebs Cycle functions only within

mitochondria.

But cytosolic isozymes of some Krebs Cycle enzymes

are involved in regenerating aspartate from fumarate.

cytosol

mitochondrial matrix

carbamoyl phosphatePi

ornithine citrulline

ornithine citrullineurea aspartate

arginine argininosuccinate

fumarate

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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.

aspartate a-ketoglutarate oxaloacetate glutamateAminotransferase (Transaminase)

COO

CH2

CH2

C

COO

O

COO

CH2

HC

COO

NH3+

COO

CH2

CH2

HC

COO

NH3+

COO

CH2

C

COO

O+ +

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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.

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Postulated mechanisms for toxicity of high [ammonia]:

1. High [NH3] would drive Glutamine Synthase:

glutamate + ATP + NH3 glutamine + ADP + Pi

This would deplete glutamatea 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 resultingdepletion ofa-ketoglutarate, an essential

Krebs Cycle intermediate, could impair energy

metabolism in the brain.

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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 a-keto acid analogs of

essential amino acids.

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

l

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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.

cytosol

mitochondrial matrix

carbamoyl phosphate

Piornithine citrulline

ornithine citrullineurea aspartate

arginine argininosuccinate

fumarate

The complete

Urea Cycle is

significantly onlyin liver.

However some

enzymes of thepathway are in

other cells and

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The amino acid arginine, in addition to being a constituent

ofproteins and an intermediate of the Urea Cycle, is

precursor for synthesis ofcreatine & the signal molecule

nitric oxide.

H3N+

C COO

CH2

CH2

CH2

NH

C

NH2

NH2

H

arginine (Arg)

H2N C N

NH2+

CH2

CH3

C

O

O

creatine

NH2 NH2 NH2

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Synthesis of the radical species nitric oxide (NO) from

arginine is catalyzed Nitric Oxide Synthase, a distant

relative of cytochrome P450.

Different isoforms of Nitric Oxide Synthase (e.g., eNOS

expressed in endothelial cells and nNOS in neuronal cells)

are subject to differing regulation.

+H3N CH COO

CH2

CH2

CH2

NH

C

NH2

NH2+

NADPH NADP+

O2 H2O O2 H2O

+H3N CH COO

CH2

CH2

CH2

NH

C

NH2

N OH

+H3N CH COO

CH2

CH2

CH2

NH

C

NH2

O

1/2NADPH 1/2NADP+

+ NO

Nitric Oxide Synthase

arginine hydroxyarginine citrulline

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NO is a short-lived signalmolecule with diverse roles

in different cell types, including regulation of smoothmuscle contraction, gene transcription, metabolism, and

neurotransmission.

Many of the regulatory effectsof NO arise from its

activation of a soluble cytosolic Guanylate Cyclase

enzyme that catalyzes synthesis ofcyclic-GMP

(analogous in structure to cyclic-AMP).

Cytotoxic effects of NO observed under someconditions are attributed to its non-enzymatic reaction

with superoxide (O2) to form the strong oxidant

peroxynitrite (ONOO).

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Polyamines include putrescine,spermidine, spermine.

Ornithine is a major precursor for

synthesis of polyamines.

Conversion of ornithine to putrescine is

catalyzed by Ornithine Decarboxylase.

+H3N CH2 CH2 CH2 CH2 NH3

+

+H3N CH2 CH2 CH2 NH CH2 CH2 CH2 CH2 NH3+

putrescine

spermidine

H3N+ C COO

CH2

CH2

CH2

NH3

H

ornithine

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The cationic polyamines have diverse roles in cell

growth & proliferation.

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

+H3N CH2 CH2 CH2 CH2 NH3+

+H3N CH2 CH2 CH2 NH CH2 CH2 CH2 CH2 NH3

+

putrescine

spermidine

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However, Ca++-activated Peptidylarginine Deiminasesconvert arginine residues within proteins to citrulline as

a post-translational modification.

H3N+ C COO

CH2

CH2

CH2

NH

C

NH2

NH2

H

H3N+ C COO

CH2

CH2

CH2

NH

C NH2

H

O

arginine citrulline

There is no tRNA for

citrulline & this amino acid

is not incorporated

translationally into proteins.

H H

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is essential to terminal differentiation of skin cells.

Excessive protein citrullination, with production of

antibodies against citrullinated proteins, is found to be

a factor in the autoimmune diseases such as rheumatoid

arthritis and multiple sclerosis.

H3N+ C COO

CH2

CH2

CH2

NH

C

NH2

NH2

H

H3N+ C COO

CH2

CH2

CH2

NH

C NH2

H

O

arginine citrulline

Substitution ofcitrulline,

which lacks arginine's

positive charge, may alter

structure & properties such as

binding affinities of a protein.

E.g., citrullination of certainproteins, including keratin

intermediate filament proteins,