FCH 532 Lecture 22 Chapter 26: Amino acid metabolism Quiz Monday on Transamination mechanism Quiz on Wed. for Urea Cycle

• • • • • • •

Urea Cycle

Excess nitrogen is excreted after the metabolic breakdown of amino acids in one of three forms: Aquatic animals are ammonotelic (release

NH 3

directly).

If water is less plentiful, NH 3 is converted to less toxic products,

urea and uric acid.

Terrestrial vertebrates are ureotelic (excrete

urea)

Birds and reptiles are uricotelic (excrete

uric acid) Urea

is made by enzymes

urea cycle

in the liver.

The overall reaction is: NH 3 + NH 3 + HCO 3 + OOC-CH 2 -CH-COO Asp O 3ATP 2ADP + 2P i + AMP + PP i NH 2 -C-NH 2 Urea + OOC-CH=CH-COO Fumarate

• • •

Urea Cycle

2 urea nitrogen atoms come from ammonia and aspartate.

Carbon atom comes from bicarbonate.

5 enzymatic reactions used, 2 in the mitochondria and 3 in the cytosol.

NH 3 + NH 3 + HCO 3 + OOC-CH 2 -CH-COO Asp O 3ATP 2ADP + 2P i + AMP + PP i NH 2 -C-NH 2 Urea + OOC-CH=CH-COO Fumarate

• 1.

2.

• •

Carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase (CPS)

catalyzes the condensation and activation NH 3 and HCO 3 -

phosphate

to form (first nitrogen containing substrate).

carbomyl

Uses 2 ATPs.

O 2ATP + NH 3 + HCO 3  NH 2 -C-OPO 3 + 2ADP + 2P i Carbamoyl phosphate

Eukaryotes have 2 types of CPS enzymes

Mitochondrial

CPSI

biosynthesis.

uses NH3 as its nitrogen donor and participates in urea Cytosolic

CPSII

uses glutamine as its nitrogen donor and is involved in pyrimidine biosynthesis.

Figure 26-8

The mechanism of action of CPS I.

• 1.

2.

3.

CPSI reaction has 3 steps

Activation of HCO3- by ATP to form

carboxyphosphate

and ADP.

Nucelophilic attack of NH3 on carboxyphosphate, displacing the phsophate to form

carbamate

and Pi.

Phosphorylation of carbamate by the second ATP to form carbamoyl phosphate and ADP The reaction is irreversible.

Allosterically activated by

N

acetylglutamate.

• • • • •

Figure 26-9

X-Ray structure of

E. coli

carbamoyl phosphate synthetase (CPS).

E. coli

has only one CPS (homology to CPS I and CPS II) Heterodimer (inactive).

Allosterically activated by ornithine (heterotetramer of (  4 ).

Small subunit hydrolyzes Gln and delivers NH 3 to large subunit.

Channels intermediate of two

reactions from one active site to the other.

• • • •

Ornithine transcarbomylase

Transfers the carbomoyl group of carbomyl phosphate to

ornithine

to make

citrulline

Reaction occurs in mitochondrion.

Ornithine produced in the cytosol enters via a specific transport system.

Citrulline is exported from the mitochondria.

• • • •

Arginocuccinate Synthetase

2nd N in urea is incorporated in the 3rd reaction of the urea cycle.

Condensation reaction with citrulline’s ureido group with an Asp amino group catalyzed by

arginosuccinate synthetase.

Ureido oxygen is activated as a leaving group through the formation of a citrulyl-AMP intermediate.

This is displaced by the Asp amino group to form arginosuccinate.

Figure 26-10

The mechanism of action of argininosuccinate synthetase.

Arigininosuccinase and Arginase

• • • • • •

Argininosuccinse

catalyzes the elimination of Arg from the the Asp carbon skeleton to form fumurate.

Arginine is the immediate precursor to urea.

Fumurate is converted by fumarase and malate dehydrogenase to to form OAA for gluconeogenesis.

Arginase

urea cycle.

ornithine.

catalyzes the fifth and final reaction of the Arginine is hydrolyzed to form urea and regenerate Ornithine is returned to the mitochondria.

1. Carbamoyl phosphate synthetase (CPS) 2. Ornithine transcarbamoylase 3. Argininosuccinate synthetase 4. Arginosuccinase 5. Arginase

Regulation of the urea cycle

• • • • •

Carbamoyl phosphate synthetase I

activated by

N-

acetylglutamate.

is allosterically

N-acetylglutamate

is synthesized from glutamate and acetyl CoA by

N-acetylglutamate synthase

, it is hydrolyzed by a specific hydrolase.

Rate of urea production is dependent on [N-acetylglutamate].

When aa breakdown rates increase, excess nitrogen must be excreted. This results in increase in Glu through transamination reactions.

Excess Glu causes an increase in

N-acetylglutamate

stimulates

CPS I

causing increases in urea cycle.

which

• • • •

Metabolic breakdown of amino acids

Degradation of amino acids converts the to TCA cycle intermediates or precursors to be metabolized to CO 2 , H 2 O, or for use in gluconeogenesis.

Aminoacids are

glucogenic, ketogenic

or both.

Glucogenic amino acids

-carbon skeletons are broken down to pyruvate,  -ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate (glucose precursors).

Ketogenic amino acids

, are broken down to acetyl-CoA or acetoacetate and therefore can be converted to fatty acids or ketone bodies.

• • • •

Metabolic breakdown of amino acids

Degradation of amino acids converts the to TCA cycle intermediates or precursors to be metabolized to CO 2 , H 2 O, or for use in gluconeogenesis.

Aminoacids are

glucogenic, ketogenic

or both.

Glucogenic amino acids

-carbon skeletons are broken down to pyruvate,  -ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate (glucose precursors).

Ketogenic amino acids

, are broken down to acetyl-CoA or acetoacetate and therefore can be converted to fatty acids or ketone bodies.

Figure 26-11

Degradation of amino acids to one of seven common metabolic intermediates.

Regulation of the urea cycle

• • • • •

Carbamoyl phosphate synthetase I

activated by

N-

acetylglutamate.

is allosterically

N-acetylglutamate

is synthesized from glutamate and acetyl CoA by

N-acetylglutamate synthase

, it is hydrolyzed by a specific hydrolase.

Rate of urea production is dependent on [N-acetylglutamate].

When aa breakdown rates increase, excess nitrogen must be excreted. This results in increase in Glu through transamination reactions.

Excess Glu causes an increase in

N-acetylglutamate

stimulates

CPS I

causing increases in urea cycle.

which

• • • •

Metabolic breakdown of amino acids

Degradation of amino acids converts the to TCA cycle intermediates or precursors to be metabolized to CO 2 , H 2 O, or for use in gluconeogenesis.

Aminoacids are

glucogenic, ketogenic

or both.

Glucogenic amino acids

-carbon skeletons are broken down to

pyruvate,



-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate (glucose precursors)

.

Ketogenic amino acids

, are broken down to

acetyl-CoA acetoacetate

and therefore can be or

converted to fatty acids or ketone bodies.

• • •

Metabolic breakdown of amino acids

Glucogenic amino acids - Ala, Ser, Cys, Gly, Met, Arg, Gln, Glu, Asn, Asp, Pro, His, Val Ketogenic amino acids - Leu, Lys Glucogenic/Ketogenic amino acids - Ile, Phe, Thr, Trp, Tyr Pathways can be organized into groups degraded into the the seven metabolic intermediates: pyruvate, oxaloacetate, a ketoglutarate, succinyl-CoA, fumarate, acetyl-CoA and acetoacetate.

Acetoacetyl-CoA can be directly converted to acetyl-CoA.

Figure 26-11

Degradation of amino acids to one of seven common metabolic intermediates.

• •

Ala, Cys, Gly, Ser, Thr are degraded to pyruvate

Trp can also be included since its breakdown product is Ala.

Alanine is converted to pyruvate through a transamination reaction which transfers the amino group to  -ketoglutarate to form glutamate and pyruvate.

1.

2.

3.

Alanine aminotransferase Serine dehydratase Glycine cleavage system 4, 5.Serine hydroxymethyl transferase 6.

7.

Threonine dehydrogenase  -amino  ketobutyrate lyase.

1.

2.

3.

Alanine aminotransferase

Serine dehydratase Glycine cleavage system

4, 5.Serine hydroxymethyl transferase 6.

7.

Threonine dehydrogenase  -amino  ketobutyrate lyase.

Serine dehydratase

• • • PLP-enzyme forms a PLP-amino acid Schiff base (like transamination) catalyzes removal of the amino acid’s  hydrogen.

Substrate loses the  -OH group undergoing an  elimination of H 2 O rather than deamination.

Aminoacrylate

, the product of this dehydration reaction, tautomerizes to the imine which hydrolyzes to pyruvate and ammonia.

Figure 26-13

The serine dehydratase reaction.

1. Formation of Ser-PLP Schiff base, 2. Removal of the  -H atom of serine, 3.  elimination of OH-, 4. Hydrolysis of Schiff base, 5. Nonenzymatic tautomerization to the imine, 6. Nonenzymatic hydrolysis to form pyruvate and ammonia.

1.

2.

3.

Alanine aminotransferase Serine dehydratase

Glycine cleavage system 4, 5.Serine hydroxymethyl transferase

6.

7.

Threonine dehydrogenase  -amino  ketobutyrate lyase.