Catabolism of proteins and amino acids

Reactions in the attachment of ubiquitin to proteins

Relationships among major pathways in nitrogen catabolism

The alpha amino acid nitrogen is channeled into glutamate.

Transaminase Roles

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).

Transaminases function in amino acid catabolism and biosynthesis

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.

a s p a r t a t e - k e t o g l u t a r a t e o x a l o a c e t a t e g l u t a m a t e

A m i n o t r a n s f e r a s e ( T r a n s a m i n a s e )

C O O

C H 2

C H 2

C

C O O

O

C O O

C H 2

HC

C O O

N H 3+

C O O

C H 2

C H 2

HC

C O O

N H 3+

C O O

C H 2

C

C O O

O + +

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

p y rid o x a l p h o sp h a te (P L P )

NH

CO

P

O O

O

O H

C H 3

CH O

H 2

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

NH

CO

P

OO

O

O

CH3

HC

H2

N

(CH2)4

Enz

H

+

RHC COO

NH2

Enzyme (Lys)-PLP Schiff base

Amino acid

The -amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP. The (+) charged N of PLP acts as an electron sink, to facilitate catalysis. Lysine extracts H+, promoting tautomerization, followed by reprotonation & hydrolysis. 

NH

CO

P

OO

O

O

CH3

HC

H2

N

HC

H

+

R COO Enz Lys NH2

Amino acid-PLP Shiff base (aldimine)

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.

NH

CO

P

O O

O

OH

CH3

CH2

NH2

H2

R C COO

O

Enz Lys NH2

Pyridoxamine phosphate (PM P)

-keto acid

Deamination of Amino Acids

Transaminases also function to 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 Dehydrogenase catalyzes the major reaction that accomplishes net removal of N from the amino acid pool. 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

+.

O O CH 2C

H 2C C C O O

O

+ N H 4+

N A D (P )+

N AD(P)H

O O CH 2C

H 2C C C O O

N H 3+

Hglu tam ate

-ke toglu tara te

G lu tam ate D ehydrogenase

H 2O

Glutamate Dehydrogenase

Summarized above: the role of transaminases in funneling amino N to glutamate, for deamination via Glutamate Dehydrogenase, producing NH4

+.

Amino acid -ketoglutarate NADH + NH4+

-keto acid glutamate NAD+ + H2O

Transaminase Glutamate Dehydrogenase

Amino Acid Oxidase

Glutamine Synthetase

Glutaminase

Muscle and liver play major roles in maintaining steady-state levels of amino acids

Glucose-Alanine Cycle

Amino acid exchange between organs

Most terrestrial land animals convert excess nitrogen to urea, a compound less toxic than ammonia, prior to excreting it.

The Urea Cycle occurs mainly in liver.

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

H 2 N C

O

N H 2

u r e a

Urea Cycle

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

Carbamoyl Phosphate Synthase is allosterically activated by N-acetylglutamate. This derivative of glutamate is synthesized when cellular [glutamate] is high, signaling excess of free amino acids due to protein breakdown or dietary intake.

H 2N C O PO 32

O

HC O 3 + N H 3 + 2 A TP

+ 2 A D P + P i

C arbam oyl Phosphate Synthase

carbam oyl phosphate

Hyperammonemia Disease

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, esp. to the brain. If not treated immediately after birth, severe mental retardation results.

Postulated mechanisms for toxicity of high [ammonia]

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.

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

would impair energy metabolism in the brain.

Hyperammonemia Disease

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.

Other disorders associated with the urea cycle

•Citrulinemia - lack of argininosuccinate synthase activity1-2 g citruline is excreted per day

•Argininosuccinicaciduria - absence of argininosuccinase activityhigh levels of argininosuccinate in blood, urine, cerebrospinal fluid

•Hyperargininemia -low levels of arginase activityelevated levels of arginine in blood and cerebrospinal fluid