Section 8. Amino Acid Metabolism

Section 8. Amino Acid Metabolism

Urea cycleUrea cycleUrea cycleUrea cycle

11/18/0511/18/05

Substrates for the Urea CycleSubstrates for the Urea Cycle

• Above, amino groups are transferred to glutamate, Above, amino groups are transferred to glutamate, from which ammonium is produced, and then used to from which ammonium is produced, and then used to make make carbamoyl phosphatecarbamoyl phosphate..

• Below, amino groups are transferred to produce Below, amino groups are transferred to produce aspartateaspartate..

• Above, amino groups are transferred to glutamate, Above, amino groups are transferred to glutamate, from which ammonium is produced, and then used to from which ammonium is produced, and then used to make make carbamoyl phosphatecarbamoyl phosphate..

• Below, amino groups are transferred to produce Below, amino groups are transferred to produce aspartateaspartate..

-amino acid

-keto acid

-ketoglutarate

glutamate NAD+

NADH

H2N-CO-OPO32-

-amino acid

-keto acid

-ketoglutarate

glutamate oxaloacetate

aspartate

1

Urea CycleUrea Cycle • Aspartate and Aspartate and carbamoyl carbamoyl phosphate phosphate each deliver an each deliver an amino group to amino group to the cycle.the cycle.

• Notice that the Notice that the carbamoyl carbamoyl phosphate phosphate production and production and condensation condensation occur in the occur in the mitochondrial mitochondrial matrix.matrix.

• Aspartate and Aspartate and carbamoyl carbamoyl phosphate phosphate each deliver an each deliver an amino group to amino group to the cycle.the cycle.

• Notice that the Notice that the carbamoyl carbamoyl phosphate phosphate production and production and condensation condensation occur in the occur in the mitochondrial mitochondrial matrix.matrix.

2

Fig. 23.16

NH4+ from Oxidative Deamination of GlutamateNH4+ from Oxidative Deamination of Glutamate

• Hexameric glutamate dehydrogenase is is controlled Hexameric glutamate dehydrogenase is is controlled allosterically.allosterically.– High energy levels inhibit (ATP and GTP).High energy levels inhibit (ATP and GTP).– Low energy levels activate (ADP and GDP).Low energy levels activate (ADP and GDP).

• NADPNADP++ can replace NAD can replace NAD++..

• NHNH44++ , which is toxic to humans, is produced in the , which is toxic to humans, is produced in the

mitochondria and used to make carbamoyl phosphate.mitochondria and used to make carbamoyl phosphate.

• Hexameric glutamate dehydrogenase is is controlled Hexameric glutamate dehydrogenase is is controlled allosterically.allosterically.– High energy levels inhibit (ATP and GTP).High energy levels inhibit (ATP and GTP).– Low energy levels activate (ADP and GDP).Low energy levels activate (ADP and GDP).

• NADPNADP++ can replace NAD can replace NAD++..

• NHNH44++ , which is toxic to humans, is produced in the , which is toxic to humans, is produced in the

mitochondria and used to make carbamoyl phosphate.mitochondria and used to make carbamoyl phosphate.

-ketoglutarateglutamate

glutamate dehydrogenase

NADH + H+ NAD+

NH4+

OO

CH2

O

CH2

O O

OO

CH2

CH2

NH3

+

O O

H

+

3

Carbamoyl Phosphate SynthesisCarbamoyl Phosphate Synthesis

• Carbamoyl phosphate synthetase is in mitochondrial matrix.Carbamoyl phosphate synthetase is in mitochondrial matrix.• NHNH44

++ is source of NH is source of NH33..• The hydrolysis of two ATP make this reaction essentially The hydrolysis of two ATP make this reaction essentially

irreversible.irreversible.• N-acetyl glutamate is an allosteric activator. (see S08L05)N-acetyl glutamate is an allosteric activator. (see S08L05)

• Carbamoyl phosphate synthetase is in mitochondrial matrix.Carbamoyl phosphate synthetase is in mitochondrial matrix.• NHNH44

++ is source of NH is source of NH33..• The hydrolysis of two ATP make this reaction essentially The hydrolysis of two ATP make this reaction essentially

irreversible.irreversible.• N-acetyl glutamate is an allosteric activator. (see S08L05)N-acetyl glutamate is an allosteric activator. (see S08L05)4

(p. 645)

1. ARGININOSUCCINATE SYNTHASE 2. ARGININOSUCCINASE1. ARGININOSUCCINATE SYNTHASE 2. ARGININOSUCCINASE3. ARGINASE 4. ORNITHINE TRANSCARBAMOYLASE3. ARGINASE 4. ORNITHINE TRANSCARBAMOYLASE1. ARGININOSUCCINATE SYNTHASE 2. ARGININOSUCCINASE1. ARGININOSUCCINATE SYNTHASE 2. ARGININOSUCCINASE3. ARGINASE 4. ORNITHINE TRANSCARBAMOYLASE3. ARGINASE 4. ORNITHINE TRANSCARBAMOYLASE

ATP

AMP + PPi

H2O

+

+

aspartate

UREA

carbamoylphosphate

fumaratearginine

argininosuccinate

ornithine

citrulline

UREACYCLE

1

23

4

2 Pi

H2N

Pi +

CH2

CH2

CH2

NNH2

NH2

+

OO

NH3

+H

H

OO

OO

H

H

CH2

CH2

CH2

NN

NH2

+

OO

NH3

+H

HH

OO

OO

H

CH2

CH2

CH2

CH2

NH3

+

OO

NH3

+H

NH2

NH2

O

O P O

O

OO

CH2

CH2

CH2

NNH2

O

OO

NH3

+H

H

OO

OO

H

CH2

NH3

+

(1-3 in cytosol, 4in mitochondial matrix)

2 ~ P used2 ~ P used

5

Connection to Krebs CycleConnection to Krebs Cycle

• Fumarate is oxidized to oxaloacetate by Krebs cycle Fumarate is oxidized to oxaloacetate by Krebs cycle enzymes, producing NADH.enzymes, producing NADH.

• Oxaloacetate accepts an amino group instead of Oxaloacetate accepts an amino group instead of being condensed with acetyl CoA.being condensed with acetyl CoA.

• Fumarate is oxidized to oxaloacetate by Krebs cycle Fumarate is oxidized to oxaloacetate by Krebs cycle enzymes, producing NADH.enzymes, producing NADH.

• Oxaloacetate accepts an amino group instead of Oxaloacetate accepts an amino group instead of being condensed with acetyl CoA.being condensed with acetyl CoA.

aspartate

fumaratearginine

argininosuccinate

citrulline

UREACYCLE

1

2

Malate

Oxaloacetate

-amino acid

-keto acid

NAD+

NADH

ATP+

AMP + PPi+

2 Pi

H20

6

Amino Acids to UreaAmino Acids to Ureaaminoacid

-ketoacid

carbamoylphosphate

ornithine UREACYCLE

glutamate

-ketoglutate

NH4+ + CO2

+ 2 ATP + H2O

2 ADP + Pi + H+ +

H2OUREA

NH2

NH2

O

*

aspartate

fumaratearginine

argininosuccinate

citrulline

Malate

Oxaloacetate

amino acid

-keto acid

AMP + PPi+

2 Pi

ATP+

**Glutamate Dehydrogenase is the control site: ADP (+), Glutamate Dehydrogenase is the control site: ADP (+), GDP (+), ATP (-), GTP (-) and NADH (-). GDP (+), ATP (-), GTP (-) and NADH (-).

Control at other sites by glucagon (+), cortisol (+), insulin Control at other sites by glucagon (+), cortisol (+), insulin (-), growth hormone (-).(-), growth hormone (-).

**Glutamate Dehydrogenase is the control site: ADP (+), Glutamate Dehydrogenase is the control site: ADP (+), GDP (+), ATP (-), GTP (-) and NADH (-). GDP (+), ATP (-), GTP (-) and NADH (-).

Control at other sites by glucagon (+), cortisol (+), insulin Control at other sites by glucagon (+), cortisol (+), insulin (-), growth hormone (-).(-), growth hormone (-).7

Summary of Reactions and Energetics - 1Summary of Reactions and Energetics - 1

HH220 + aa + NAD0 + aa + NAD++ -keto acid + NH-keto acid + NH44++ + NADH + H + NADH + H++

and and

HH220 + fumarate + aa + NAD0 + fumarate + aa + NAD++ aspartate + aspartate + -keto acid -keto acid + NADH + H+ NADH + H++

then then

aspartate + NHaspartate + NH44++ + HCO + HCO33

-- + 3 ATP + 3 ATP urea + fumarate + 2 Hurea + fumarate + 2 H220 + 2 ADP + AMP + 4 Pi + H0 + 2 ADP + AMP + 4 Pi + H+ +

Four high energy phosphate bond equivalents are used Four high energy phosphate bond equivalents are used for these reactions (- 4 ~P).for these reactions (- 4 ~P).Two NADH are produced.Two NADH are produced.

HH220 + aa + NAD0 + aa + NAD++ -keto acid + NH-keto acid + NH44++ + NADH + H + NADH + H++

and and

HH220 + fumarate + aa + NAD0 + fumarate + aa + NAD++ aspartate + aspartate + -keto acid -keto acid + NADH + H+ NADH + H++

then then

aspartate + NHaspartate + NH44++ + HCO + HCO33

-- + 3 ATP + 3 ATP urea + fumarate + 2 Hurea + fumarate + 2 H220 + 2 ADP + AMP + 4 Pi + H0 + 2 ADP + AMP + 4 Pi + H+ +

Four high energy phosphate bond equivalents are used Four high energy phosphate bond equivalents are used for these reactions (- 4 ~P).for these reactions (- 4 ~P).Two NADH are produced.Two NADH are produced.

8

Summary of Reactions and Energetics - 2Summary of Reactions and Energetics - 2

Now consider NADH oxidation:Now consider NADH oxidation:

2 H2 H++ + 2 NADH + O + 2 NADH + O22 2 NAD2 NAD++ + 2 H + 2 H220 (+5 ~P)0 (+5 ~P)

The net reaction is thenThe net reaction is then

2 aa + HCO2 aa + HCO33-- + O + O22

2 2 -keto acid + urea + H-keto acid + urea + H++ + 2 H + 2 H220 (+1~P) 0 (+1~P)

Now consider NADH oxidation:Now consider NADH oxidation:

2 H2 H++ + 2 NADH + O + 2 NADH + O22 2 NAD2 NAD++ + 2 H + 2 H220 (+5 ~P)0 (+5 ~P)

The net reaction is thenThe net reaction is then

2 aa + HCO2 aa + HCO33-- + O + O22

2 2 -keto acid + urea + H-keto acid + urea + H++ + 2 H + 2 H220 (+1~P) 0 (+1~P)

9

HyperammonemiaHyperammonemia

• Normal blood [NHNormal blood [NH44++] is 10-40 ] is 10-40 M.M.

• Deficiencies of carbamoyl phosphate synthetase or Deficiencies of carbamoyl phosphate synthetase or of any enzyme in the urea cycle cause high [NHof any enzyme in the urea cycle cause high [NH44

++].].

• Affects CNS and can lead to irreversible brain Affects CNS and can lead to irreversible brain damage.damage.

• Treatment strategies depend on which enzyme is Treatment strategies depend on which enzyme is deficient.deficient.

• Normal blood [NHNormal blood [NH44++] is 10-40 ] is 10-40 M.M.

• Deficiencies of carbamoyl phosphate synthetase or Deficiencies of carbamoyl phosphate synthetase or of any enzyme in the urea cycle cause high [NHof any enzyme in the urea cycle cause high [NH44

++].].

• Affects CNS and can lead to irreversible brain Affects CNS and can lead to irreversible brain damage.damage.

• Treatment strategies depend on which enzyme is Treatment strategies depend on which enzyme is deficient.deficient.

10

Argininosuccinase DeficiencyArgininosuccinase Deficiency• Low dietary protein Low dietary protein reduces need for urea reduces need for urea cycle.cycle.

• Low dietary protein Low dietary protein reduces need for urea reduces need for urea cycle.cycle.

• High dietary arginine High dietary arginine provides a path for provides a path for carbamoyl phosphate carbamoyl phosphate and aspartate and aspartate nitrogens to produce nitrogens to produce argininosuccinate, argininosuccinate, which is excreted.which is excreted.

• High dietary arginine High dietary arginine provides a path for provides a path for carbamoyl phosphate carbamoyl phosphate and aspartate and aspartate nitrogens to produce nitrogens to produce argininosuccinate, argininosuccinate, which is excreted.which is excreted.

ATP

AMP + PPi

H2O

+

+

aspartate

UREA

carbamoylphosphate

fumarate

arginine

argininosuccinate

ornithine

citrulline

UREACYCLE

1

23

4

2 Pi

H2N

CH2

CH2

CH2

NNH2

NH2

+

OO

NH3

+H

H

OO

OO

H

H

CH2

CH2

CH2

NN

NH2

+

OO

NH3

+H

HH

OO

OO

H

CH2

CH2

CH2

CH2

NH3

+

OO

NH3

+H

NH2

NH2

O

O P O

O

OO

CH2

CH2

CH2

NNH2

O

OO

NH3

+H

H

OO

OO

H

CH2

NH3

+

(excreted)

(excess supplied)11

Carbamoyl Phosphate Synthetase DeficiencyCarbamoyl Phosphate Synthetase Deficiency

• Hippurate and phenylacetylglutamine are excreted.Hippurate and phenylacetylglutamine are excreted.• Amino groups to glycine and glutamine by transamination.Amino groups to glycine and glutamine by transamination.

• Hippurate and phenylacetylglutamine are excreted.Hippurate and phenylacetylglutamine are excreted.• Amino groups to glycine and glutamine by transamination.Amino groups to glycine and glutamine by transamination.

12

Fig. 23.20

Ketogenic and Glucogenic Amino AcidsKetogenic and Glucogenic Amino Acids

• After removal of the amino group, the keto acids are used to make After removal of the amino group, the keto acids are used to make Krebs cycle intermediates, pyruvate, acetyl CoA and acetoacetyl CoA.Krebs cycle intermediates, pyruvate, acetyl CoA and acetoacetyl CoA.

• After removal of the amino group, the keto acids are used to make After removal of the amino group, the keto acids are used to make Krebs cycle intermediates, pyruvate, acetyl CoA and acetoacetyl CoA.Krebs cycle intermediates, pyruvate, acetyl CoA and acetoacetyl CoA.13

Fig. 23.21

Nitrogen for Oral BacteriaNitrogen for Oral Bacteria

• Urea is a major source of nitrogen for oral Urea is a major source of nitrogen for oral bacteria.bacteria.

• It diffuses through most membranes and is It diffuses through most membranes and is in saliva.in saliva.

• Bacterial urease produces NHBacterial urease produces NH44++..

• Glutamate dehydrogenase incorporates Glutamate dehydrogenase incorporates NHNH44

+ + into into -keto acids to obtain-keto acids to obtainamino amino

acids for bacterial growth.acids for bacterial growth.

• Urea is a major source of nitrogen for oral Urea is a major source of nitrogen for oral bacteria.bacteria.

• It diffuses through most membranes and is It diffuses through most membranes and is in saliva.in saliva.

• Bacterial urease produces NHBacterial urease produces NH44++..

• Glutamate dehydrogenase incorporates Glutamate dehydrogenase incorporates NHNH44

+ + into into -keto acids to obtain-keto acids to obtainamino amino

acids for bacterial growth.acids for bacterial growth.

H2NCONH2 + H+ + H2O 2 NH4+ + HCO3

-urease

14

Nitrogen for Bacterial Amino Acid SynthesisNitrogen for Bacterial Amino Acid Synthesis

glutamate

+ NH4+ glutamine

glutamine synthase

ATP ADP + Pi

-ketoglutarate+ glutamine 2 glutamate

glutamate synthase

NADPH + H+ NADP+

-ketoglutarate

+ NH4+

glutamate glutamate

dehydrogenase

NADPH + H+ NADP+

+ H20

• When [NHWhen [NH44++] is limiting, it does not bind glutamate dehydrogenase, and the ] is limiting, it does not bind glutamate dehydrogenase, and the

lower two reactions are used.lower two reactions are used.• When [NHWhen [NH44

++] is limiting, it does not bind glutamate dehydrogenase, and the ] is limiting, it does not bind glutamate dehydrogenase, and the lower two reactions are used.lower two reactions are used.15

Engineered Oral Bacteria to Fight Caries?Engineered Oral Bacteria to Fight Caries?

• Streptococcus Salivarius urease activity affects Streptococcus Salivarius urease activity affects oral microbial ecology.oral microbial ecology.

• It produces NHIt produces NH33, which in addition to promoting , which in addition to promoting

growth, neutralizes acids produces by other growth, neutralizes acids produces by other bacteria.bacteria.

• S. Salivarius urease gene was introduced into S. Salivarius urease gene was introduced into Streptococcus mutans GS5. It was expressed Streptococcus mutans GS5. It was expressed and during glucose metabolism reduced pH and during glucose metabolism reduced pH decrease and duration.decrease and duration.

• (Clancy & Burne,1997 FEMS Microbiol Lett 151:205)(Clancy & Burne,1997 FEMS Microbiol Lett 151:205)

• Streptococcus Salivarius urease activity affects Streptococcus Salivarius urease activity affects oral microbial ecology.oral microbial ecology.

• It produces NHIt produces NH33, which in addition to promoting , which in addition to promoting

growth, neutralizes acids produces by other growth, neutralizes acids produces by other bacteria.bacteria.

• S. Salivarius urease gene was introduced into S. Salivarius urease gene was introduced into Streptococcus mutans GS5. It was expressed Streptococcus mutans GS5. It was expressed and during glucose metabolism reduced pH and during glucose metabolism reduced pH decrease and duration.decrease and duration.

• (Clancy & Burne,1997 FEMS Microbiol Lett 151:205)(Clancy & Burne,1997 FEMS Microbiol Lett 151:205)

16

Web linksWeb links

Nitrogen Fixation. A summary of the topic.. A summary of the topic.

Nitrogen Cycle. The biological big picture.. The biological big picture.

Amino Acid Metabolism. Reviews reactions.. Reviews reactions.

Next topic:Next topic: Porphyrins, heme, Porphyrins, heme, bile pigmentsbile pigments

Web linksWeb links

Nitrogen Fixation. A summary of the topic.. A summary of the topic.

Nitrogen Cycle. The biological big picture.. The biological big picture.

Amino Acid Metabolism. Reviews reactions.. Reviews reactions.

Next topic:Next topic: Porphyrins, heme, Porphyrins, heme, bile pigmentsbile pigments