Molecular Biochemistry II

Amino Acid Catabolism: N

Copyright 1999-2008 by Joyce J. Diwan.

All rights reserved.

H
R1 C COO

R2

Transaminases
(aminotransferases)
catalyze the
reversible reaction
at right.

NH3

C COO

O
Transaminase
H

R1 C COO
O

R2

C COO

NH3

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

COO

COO

CH2

COO

CH2

CH2

CH2

CH2

CH2

HC

NH3+

COO

COO

COO

HC

NH3+

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.

CH3
HC

COO

COO

CH2

CH2

CH2
NH3+

COO

alanine

CH3
O

COO

-ketoglutarate

CH2
O

COO

pyruvate

HC

NH3+

COO

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

H2
C

O
OH

N
H

CH3

pyridoxal phosphate (PLP)

The prosthetic group of Transaminase is

pyridoxal phosphate (PLP), a derivative of
vitamin B6.

H
C

COO

Enz
(CH2)4

NH2

Amino acid
O

P
O

HC
H2
C

N+

H
O

N
H

CH3

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

The -amino group

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

P
O

R
HC

H2
C

H
C
N+

COO
H
O

N
H

CH3

Amino acid-PLP Shiff base (aldimine)

The active site lysine extracts H+, promoting

tautomerization, followed by reprotonation & hydrolysis.

O
EnzLysNH2
O

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

P
O

H2
C

NH2
CH2

COO
-keto acid
C

OH

N
CH3
H
Pyridoxamine phosphate (PMP)

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.

EnzLysNH2

P
O

R
HC

H2
C

H
C
N+

COO
H
O

N
H

CH3

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.

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 of -ketoglutarate, at the active site.
Students should then show and explain the structure
displayed by them to the neighboring student or
student group.

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.

NH3+

H2 H2
OOC C C C
glutamate
H

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

H2O

COO
NAD(P)+
NAD(P)H

H2 H2
OOC C C C
-ketoglutarate

COO + NH4+

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+.

Amino acid

-ketoglutarate

-keto acid

glutamate

Transaminase

NADH + NH4+

NAD+ + H2O

Glutamate
Dehydrogenase

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

+
H2O NH4

H2O
HO

CH2

H
C

COO

NH3+

serine

H2C

COO

NH3+

aminoacrylate

H3C

COO

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
Catalase catalyzes: 2 H2O2 2 H2O + O2

O
H2N

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.

HCO3

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

ATP
ADP
O
HO
NH3
Pi

Ammonia is the N input.

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

H2N

OPO32

carbonyl phosphate

O
C

ATP
ADP

carbamate

O
H2N

OPO32

carbamoyl phosphate

HCO3

Alternate forms of
Carbamoyl Phosphate
Synthase (Types II & III)
initially generate ammonia
by hydrolysis of glutamine.
The type II enzyme includes
a long internal tunnel
through which ammonia &
reaction intermediates such
as carbamate pass from one
active site to another.

ATP
ADP
O
HO
NH3
Pi
H2N

OPO32

carbonyl phosphate

O
C

ATP
ADP

carbamate

O
H2N

OPO32

carbamoyl phosphate

HCO3 + NH3 + 2 ATP

O
H2N

Carbamoyl Phosphate
Synthase
OPO32 + 2 ADP + Pi

carbamoyl phosphate

Carbamoyl Phosphate Synthase is the committed step of

the Urea Cycle, and is subject to regulation.

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
NH3+

Urea Cycle

CH2

NH3+

COO

ornithine
4

O
H2N

urea

NH

NH2

H2O

CH2

COO
CH2

HC

H
N

COO
COO

CH2

HC

CH2
HC

ATP
AMP + PPi

NH

arginine

CH
NH3+

COO

CH2

COO

CH2

COO

NH3+

HC

NH2+

H2N

citrulline

CH2

Pi

Urea Cycle

NH2

CH2

CH2
HC

OPO32

carbamoyl
phosphate

CH2

Enzymes in
mitochondria:
1. Ornithine
Transcarbamylase
Enzymes in
cytosol:
2. ArgininoSuccinate
Synthase
3. Argininosuccinase
4. Arginase.

H2N

COO

fumarate

HC

NH2

COO

aspartate
C

NH2+

NH
CH2
CH2

argininosuccinate

CH2
HC

NH3+

COO

cytosol
mitochondrial matrix
carbamoyl phosphate
Pi
ornithine
ornithine
urea
arginine

citrulline
citrulline
aspartate
argininosuccinate

fumarate

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
ornithine
urea
arginine

citrulline
citrulline
aspartate
argininosuccinate

fumarate

A complete Krebs Cycle functions only within

mitochondria.
But cytosolic isozymes of some Krebs Cycle enzymes are
involved in regenerating aspartate from fumarate.

COO

COO

COO

CH2

COO

CH2

CH2

CH2

CH2

CH2

HC

NH3+

COO

COO

COO

HC

NH3+

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.

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

The complete
Urea Cycle is
significantly only
in liver.

mitochondrial matrix
carbamoyl phosphate
Pi
ornithine
citrulline

However some
ornithine
citrulline
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.

CH3
H2N

O
CH2

NH2+

C
O

creatine

The amino acid arginine, in addition to being a constituent

of proteins and an intermediate of the Urea Cycle, is
precursor for synthesis of creatine & the signal molecule
nitric oxide.

NH2
C

NH2

NH

C
NADPH

NADP+

CH2
O2

CH

OH

CH2
O2

arginine

H3 N

CH

NO

CH2

H2O

CH2

CH2
COO

NH

1/2 NADPH 1/2 NADP+

CH2

H2O

CH2
H3N

NH

CH2

CH2

NH2

NH2

COO

hydroxyarginine

H3N

CH

COO

citrulline

Nitric Oxide Synthase

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.

 NO is a short-lived signal molecule with diverse roles

in different cell types, including regulation of smooth
muscle contraction, gene transcription, metabolism, and
neurotransmission.
Many of the regulatory effects of NO arise from its
activation of a soluble cytosolic Guanylate Cyclase
enzyme that catalyzes synthesis of cyclic-GMP
(analogous in structure to cyclic-AMP).
Cytotoxic effects of NO observed under some
conditions are attributed to its non-enzymatic reaction
with superoxide (O2) to form the strong oxidant
peroxynitrite (ONOO).

 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.

There is no tRNA for

citrulline & this amino acid
is not incorporated
translationally into proteins.

However, Ca++-activated Peptidylarginine Deiminases

convert arginine residues within proteins to citrulline as
a post-translational modification.

Substitution of citrulline,
which lacks arginine's
positive charge, may alter
structure & properties such as
binding affinities of a protein.
E.g., citrullination of certain
proteins, including keratin
intermediate filament proteins,
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.