Aino Acid Catabolism

8/6/2019 Aino Acid Catabolism

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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 E-k eto acid acceptors.

H

R1 C COO-

+ R2 C COO-

NH3

+

O

Transaminase

H

R1 C COO

-

+ R2 C COO

-

O NH3

+

Transaminases

(aminotransferases)catalyze thereversible reactionat right.



Example of a Transaminase reaction:

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

E-Ketoglutarate accepts the amino group,

becoming the amino acid glutamate.

aspartate E-ketoglutarate oxaloacetate glutamate

Aminotrans erase (Transaminase)

COO

CH2

CH2

C

COO

O

COO

CH2

HC

COO

NH3+

COO

CH2

CH2

HC

COO

NH3+

COO

CH2

C

COO

O+ +



In another example, alanine becomes pyruvate as

the amino group is transferred to E-ketoglutarate.

alanine E-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+ +



Transaminases equilibrate amino groups amongavailableE-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 aresynthesized.

Although the amino N of one amino acid can be usedto 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 (E-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.)



The prosthetic group of Transaminase is

pyridoxal phosphate (PLP), a derivative of

vitamin B6.

pyridoxal phosphate (PLP)

NH

C

O

P

O

O

O

OH

CH3

C

H O

H2



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

NH

C

O

P

O

O

O

O

CH3

HC

H2

N

(CH2)4

Enz

H

+

RHC COO

NH2

Enzyme (Lys)-PLP Schiff base

Amino acid





The amino group remains on what is now pyridoxamine

phosphate (PMP).

A different E-keto acid reacts with PMP and the processreverses, to complete the reaction.

N

H

C

O

P

OO

O

OH

CH3

CH2

NH2

H2

R C COO

O

Enz sNH2

Pyridoxamine phosphate (PMP)

E-keto acid

What was anamino acidleaves as anE-k eto acid.



Several other enzymes that catalyze metabolism or synthesis of amino acids also utilize PLP as prostheticgroup, and have mechanisms involving a Schiff base

linkage of the amino group to PLP.

NH

C

O

P

OO

O

O

CH3

HC

H2

N

HC

H+

R COO

Enz sNH2

Amino acid- hi base (aldimine)



Chime Exercise

Two neighboring students or student groups shouldteam up, each displaying one of the following:

Transaminase with PLP in Schiff base linkage tothe active site lysine residue.

Transaminase in the PMP form, with glutarate, ananalog of E-ketoglutarate, at the active site.

Students should then show and ex plain the structuredisplayed by them to the neighboring student or student group.



In addition to equilibrating amino groups among

available E-keto acids, transaminases funnel amino

groups from excess dietary amino acids to those amino

acids (e.g., glutamate) that can be deaminated.

Carbon sk eletons 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.



It is one of the few enzymes that can use NAD+ or NADP+

as e acceptor.

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

+.

OOCH

2CH

2C C COO

O

+ NH4+

NAD(P)+

NAD(P)H

OOC

H2C

H2C C COO

NH3+

Hglutamate

E-ketoglutarate

Glutamate ehydrogenase

H2O

Glutamate

Dehydrogenase

catalyzes a major reaction that effects

net removal of Nfrom the aminoacid pool.



Summarized above:The role of transaminases in funneling amino N toglutamate, which is deaminated via lutamateDehydrogenase, producing NH4

+.

Amino acid E-ketoglutarate NADH + NH4+

E-keto acid glutamate NAD+

+ H2O

Transaminase Glutamate Dehydrogenase



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 E-k eto acid + NH4

+ + FADH2

FADH2 + O2 FAD + H2O2

Catalase catalyzes: 2H2O22 H2O + O2

HO CH2

HC COO

NH3+

C COO

OH2O NH4+

C COO

NH3+

H2C H3C

H2O

serine aminoacrylate pyruvate

Serine Dehydratase



Most terrestrial land animals convert excess nitrogen tourea, 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 lutamate 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



Carbamoyl PhosphateSynthase (Type I) catalyzes

a 3-step reaction, with

carbonyl phosphate and

carbamate intermediates.Ammonia is the N input.

The reaction, which

involves cleavage of 2 ~P bonds of ATP, is essentially

irreversible.

H2N C OPO32

O

H2N C O

O

HO C

O

OPO32

HCO3

ATP

NH3

ADP

ATP

Pi

ADP

carbonyl phosphate

carbamate

carbamoyl phosphate



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.

H2N C OPO32

O

H2N C O

O

HO C

O

OPO32

HCO3

ATP

NH3

ADP

ATP

Pi

ADP

carbonyl phosphate

carbamate

carbamoyl phosphate



Carbamoyl Phosphate Synthase is the committed step

of the Urea Cycle, and is subject to regulation.

H2N C OPO

3

2

O

HCO3

+ NH3 + 2 ATP

+ 2 ADP + Pi

Carbamoyl hosphateynthase

carbamoyl phosphate



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

This derivative of glutamate is synthesized fromacetyl-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)

N

H

C COO

CH2

CH2

COO

H

CH3C

O

N -acet lgluta ate



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

umarate

arginine

carbamoyl phosphate

rea 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.



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

mitochon rial matri

carbamoyl phosphatei

ornithine citrulline

ornithine citrullineurea aspartate

arginine argininosuccinate

fumarate



A complete K rebs Cycle functions only withinmitochondria.

But cytosolic isozymes of some Krebs Cycle enzymes

are involved in regenerating aspartate from fumarate.

cytosol

mitoc o rial matri

carbamoyl phosphate

i


ornithine citrullineurea aspartate

arginine argininosuccinate

fumarate



Fumarate is converted to oxaloacetate via Krebs Cycle

enzymes Fumarase & Malate Dehydrogenase.Oxaloacetate is converted to aspartate viatransamination (e.g., from glutamate).

Aspartate then reenters Urea Cycle, carrying an amino

group derived from another amino acid.

aspartate E-ketoglutarate oxaloacetate glutamate

Aminotrans erase (Transaminase)

COO

CH2

CH2

C

COO

O

COO

CH2

HC

COO

NH3+

COO

CH2

CH2

HC

COO

NH3+

COO

CH2

C

COO

O+ +



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 driveG

lutamine Synthase:glutamate + ATP + NH3 glutamine + ADP + Pi

This would deplete glutamate ± a neurotransmitter &

precursor for synthesis of the neurotransmitter ABA.

2. Depletion of glutamate & high ammonia level would

drive Glutamate Dehydrogenase reaction to reverse:

glutamate + NAD(P)+ E-k etoglutarate +

NAD(P)H + NH4+

The resulting depletion of E-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 inta

k e to the amount barelyadequate to supply amino acids for growth, while

adding to the diet the E-keto acid analogs of essential amino acids.

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



tissues where they generate arginine & ornithine, which

are precursors for other important molecules.E.g., Argininosuccinate Synthase, which catalyzessynthesis 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

Pi



urea aspartatearginine argininosuccinate

fumarate

The complete

Urea Cycle issignificantly onlyin liver.

However some

enzymes of the pathway are inother cells and



The amino acid arginine, in addition to being a constituentof proteins and an intermediate of the Urea Cycle, is precursor for synthesis of creatine & the signal moleculenitric oxide.

H3

H2

H2

H2

H

C

NH2

NH2

H

a e (

H2N C N

NH2+

CH2

CH3

C

O

O

creatine



Synthesis of the radical species nitric oxide (·NO) fromarginine is catalyzedNitric Oxide Synthase, a distantrelative of cytochrome P450.

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

ex pressed 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

1/2 NADPH 1/2 NADP+

+ NO

Nitric Oxide Synthasearginine hydroxyarginine citrulline



·NO is a short-lived signal molecule with diverse rolesin different cell types, including regulation of smoothmuscle contraction, gene transcription, metabolism, andneurotransmission.

Many of the regulatory effects of ·NO arise from itsactivation 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 someconditions are attributed to its non-enzymatic reactionwith superoxide (O2·

) to form the strong oxidantperoxynitrite (ONOO).



Polyamines include putrescine,spermidine, spermine.

Ornithine is a major precursor for synthesis of polyamines.

Conversion of ornithine to putrescine iscatalyzed by Ornithine Decarboxylase.

+

H3N CH2 CH2 CH2 CH2 NH3+

+H3N CH2 CH2

CH2 NH CH2CH2 CH2

CH2 NH3+

putr escine

s per idine

H3N+

C COO

CH2

CH2

CH2

NH3

H

ornithine



The cationic polyamines have diverse roles in cellgrowth & proliferation.

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

+

3 2 2 2 2 3+

+3 2 2 2 2 2 2 2 3

+

putrescine

s per i ine



However, Ca++-activated PeptidylarginineDeiminasesconvert arginine residues within proteins to citrulline asa 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

arginin itrullin

Th r is no tRNA for citrulline & this amino acid

is not incorpor at dtr anslationally into prot ins.



is essential to terminal differentiation of sk in cells.

Excessive protein citrullination, with production of antibodies against citrullinated proteins, is found to bea factor in the autoimmune diseases such as rheumatoidarthritis and multiple sclerosis.

H N C COO

CH

CH

CH

NH

C

NH

NH

H

H N C COO

CH

CH

CH

NH

C NH

H

O

ar i i citr lli

S bstit tio of citrulline,

which lack s ar i i 'spositive charge, may alt r str ct r & prop rti s s ch as bi di affi iti s of a prot i .

E. ., citrullination of certai protei s, i cl di k eratin

i termediate filament proteins,

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Aino Acid Catabolism