nitrogen metabolism

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Nitrogen Metabolism • Protein degradation and turnover • Amino acid degradation and urea cycle • Nitrogen cycle • Nitrogen fixation • Amino acid biosynthesis • Amino acid derivatives

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Nitrogen Metabolism. Protein degradation and turnover Amino acid degradation and urea cycle Nitrogen cycle Nitrogen fixation Amino acid biosynthesis Amino acid derivatives. How Much Protein?. A 70 kg person (154 lb) typically consumes 100 g protein per day - PowerPoint PPT Presentation

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Nitrogen Metabolism

• Protein degradation and turnover

• Amino acid degradation and urea cycle

• Nitrogen cycle

• Nitrogen fixation

• Amino acid biosynthesis

• Amino acid derivatives

How Much Protein?

• A 70 kg person (154 lb) typically consumes 100 g protein per day

• To stay in nitrogen balance that person must excrete 100 g of N products per day

• The body makes 400 g of protein per day and 400 g are broken down

• 300 g of amino acids recycled into new protein, 100 g are degraded

• Total protein = 500 g/day, 400 g degraded, 400 resynthesized and 100 g catabolized

Characteristic of Proteins in Cells

• Synthesized and degraded constantly -Turnover

• Turnover may be minutes, weeks or longer• Synthesis requires essential and non essential

amino acids• Degradation is programmed and regulated• Control point enzymes most labile; constitutive

most stable• Nutritional state and hormones affect

degradation rates (glucocorticoids, insulin, etc.)

The half-life of proteins is determined by rates of synthesis and degradation

A given protein is synthesized at a constant rate KS

A constant fraction of active molecules are destroyed per unit time

C is the amount of Protein at any time

KD is the first order rate constant of enzyme degradation, i.e., the fraction destroyed per unit time, also depends on the particular protein

KS is the rate constant for protein synthesis; will vary depending on the particular protein

Rate of Turnover = dC

dt = KS - KDC

Steady-state is achieved when the amount of protein synthesized per unit time equals the amount being destroyed

dC

dt= 0 KDC = KS t 1/2 =

0.693

KD

Proteinconcentration(enzyme activity)

Hours after stopping synthesis

C

Stop protein synthesis,measure rate of decay

Steps in Protein Degradation

Transformation to a degradable form(Metal oxidized, Ubiquination, N-terminal residues, PEST sequences)

Lysosomal DigestionLysosomal Digestion 26S Proteasome digestion26S Proteasome digestion

Proteolysis to peptides

UbiquinationUbiquination

ATP

AMP + PPi

KFERQ8 residue fragments

7 type, 7 type subunits

N-end rule: DRLKF: 2-3 min AGMSV: > 20 hrPEST: Rapid degradation

COO-Ubiquitin

Glycine at C terminal of Ubiquitin

SC

O

E1

HSATP

AMP + PPi

E1

HS

HS E1

E2H3N+

NH3+

NH3+

N

NCO

N CO

CO

ATP

AMP + PPi

Ubiquitin-specific proteases(26S proteasome)

Degradedprotein + Ubiquitin

Ubiquination

Ubiquitin activating enzyme

Activationof Ubiquitin

Ubiquitin conjugating enzyme 20 or more per cell

SC

O

E23

E2 SH3

Ubiquitin ligase

E3

Page 1075

CPoly Ubiquitin

NH

O

Cervical Cancer

Human Papilloma virus (HPV)

Activates the E3 that catalyzes ubiquination of p53 tumor suppressor and DNA repair enzymes

(occurs in 90% of cervical cancers)

Mutated DNA is unchecked and allowed to replicate

P472

26S Proteasome (2000 kD)

Opening for ubiquinated protein to enter

20S

19S

19S

7 alpha7 betaSubunits

Catalysis in beta

8-residue peptides diffuse out

Amino Acids

Amine Group

Glutamate

Urea

Carbon Skeleton

DegradationBiosynthesis

CO2 + H2OAmino Acids

Amino Acid Derivatives

COO-

C=O

CH2

COO-

CH2

H3N-C-H

COO-

CH2

COO-

CH2

+

-Ketoglutarate-Glutamate

Amine group acceptor

Amine group donor

AA1 + -KG -ketoacid + glutamate

Amino transferases

Requires pyridoxal-5’-phosphate

-Kg L-glutamate

acceptor donor

N

CH2OPHO

H3C

CH

O

N

CH2OHHO

H3C

CH2OHVitamin B6

Pyridoxine

Cofactor (N acceptor)

Pyridoxal-5’-PO4

N

CH2OPHO

H3C

CH2NH2Cofactor (N donor)Pyridoxamine-PO4

Alanine-Pyruvate Aminotransferase

COO-

C=O

CH2

COO-

CH2

+

COO-

H3N-C-H

CH3

+

N

CH2OPHO

H3C

CH

O

+H3N-C-H

COO-

CH2

COO-

CH2

+COO-

CH3

C=O

N

CH2OPHO

H3C

CH2NH2

N

CH2OPHO

H3C

CH

Oforward reverse

Alanine

Enz-CHO(E-B6-al)

Enz-NH2

(E-B6-am)

Pyruvate -Ketoglutarate

Enz-NH2 Enz-CHO

Glutamate

Ordered Ping-Pong Mechanism

Mechanism

In Out In Out

Glutamate Metabolism

COO-

C=O

CH2

COO-

CH2

+ NAD(P)+

+ NH4+

+ H2O + NAD(P)H + H+

Glutamate dehydrogenase

Urea cycle

specific for glutamate

requires NAD+

Forward Reaction

delivers NH4+ to urea cycle

Reverse Reaction

specific for -ketoglutarate

requires NADPH

Fixes NH4+, prevents toxicity

H3N-C-H

COO-

CH2

COO-

CH2

+

Glutamine Metabolism

H3N-C-H

COO-

CH2

COO-

CH2

+

+ ADP + Pi

Glutamine Synthetase

H3N-C-H

COO-

CH2

COO-

CH2

+

Glutaminase

+ NH4+

H2O

H3N-C-H

COO-

CH2

CH2

+

C=OOPO3

=

+ ATP + NH4+

H3N-C-H

COO-

CH2

CH2

+

C=ONH2

L-glutamine

intermediate

Glutamate-PO4

Urea

Overall Scheme Using Alanine as an Example

Alanine Pyruvate

-ketoglutarate glutamate

NH4+

Urea

Amino transferase with pyridoxal-5’-PO4

Glutamate dehydrogenase with NAD+

Glutaminase with H2O

glutamine

Glutamate and glutamine are the only donors of NH3 to the Urea Cycle

The Urea Cycle

1. Occurs in the liver mitochondria and cytosol

2. Starts with carbamoyl-PO4

3. Ends with arginine

4. Requires aspartate

5. Requires 3 ATPs to make one urea

NH4+ + HCO3

- + 2 ATP

Synthesis of Carbamoyl-PO4

H2NC

O

O-P-O

O

O

~

High energy bond

+ 2 ADP + Pi

Carbamoyl phosphate Synthetase ICarbamoyl phosphate Synthetase I

Ornithine

Citrulline

Argininosuccinate

Arginine

Carbamoyl-PO4

Aspartate

Urea

ATP

Urea Cycle

Urea Cycle

H2ONH

CH2

CH2

CH2

COO-

CH3N

H

H2N=CNH2+

NH3

CH2

CH2

CH2

COO-

CH3N

H

+

C

H2N NH2

O

COPO3H2N

O

COO-

CH2

H3N+-C-H

NH3

CH2

CH2

+

+

O=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

+ OPO3=

Ornithine

Carbamoyl-PO4Citruline

Reactions of Urea Cycle

O=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

+

COO-

CH2

COO-

H-C-NH3

+

=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

COO-

CH2

COO-

H-C-NL-Aspartate

Argininosuccinate

ATP ADP + Pi

Mitochondria

Cytosol

=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

COO-

CH2

COO-

H-C-N =C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

H2N+

COO-

COO-

CH2

C-OHH

+

COO-

COO-

C

C

H

H

COO-

COO-

CH2

C=O

COO-

CH2

COO-

H-C-NH3

+

Fumarate

L-MalateOxaloacetateL-Aspartate

Cytosol

L-Arginine

=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

H2N+

COO-

CH2

H3N+-C-H

NH3

CH2

CH2

+

H2NC

O

NH2

Urea

+

Ornithine

L-Arginine

H2O

Return to Mitochondria