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Nitrogen Assimilation. How is NH 3 incorporated into organic molecules? Glutamate Dehydrogenase vs Glutamate Synthase Properties of Glutamine Synthetase Regulation of Glutamine Synthetase Glutamine as a major nitrogen donor. CO 2. ATP. NH 3. Carbamoyl phosphate. Glutamate.

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Nitrogen assimilation
Nitrogen Assimilation

  • How is NH3 incorporated into organic molecules?

  • Glutamate Dehydrogenase vs Glutamate Synthase

  • Properties of Glutamine Synthetase

  • Regulation of Glutamine Synthetase

  • Glutamine as a major nitrogen donor


CO2

ATP

NH3

Carbamoyl

phosphate

Glutamate

Glutamine

Asparagine

None

Other

amino

acids

Purine nucleotides,

Cytidine nucleotides

Amino sugars,

Tryptophan, Histidine

Pyrimidine nucleotides

Arginine

Urea

The only inorganic

nitrogen source for

mammals is NH3

3 Gateways to

Biological Molecules

a-Ketoglutarate

Aspartate

Glutamate

Of the 3, the most versatile is glutamine


Glutamate Dehydrogenase

-Kg + NH3 + NAD(P)H + 2H+

Glutamate + NAD(P)+

+ H2O

Bacteria and Plants

Make glutamate, assimilate NH3

Glutamate is major solute in the

bacteria

Provide NH3 to urea cycle,

provide -Kg to Krebs

Animals

High Km for NH3 limits forward

Mitochondrial location

(-) by ATP, GTP

(+) by ADP, GDP


Glutamate Synthase (Bacteria Only)

-Kg + glutamine + NADPH + H+

2 glutamates + NADP+

Glutamine is the nitrogen donor

Reaction is a reductive amination


Each subunits subject to allosteric

regulation

Glutamine Synthetase is a Primary Regulatory

Point in Nitrogen Metabolism

Properties of Bacterial Enzyme

12 identical 50,000 mwt subunits

6

Combined Mwt of 600,000

6

Hexagonal stacked rings

SIDE VIEW

8 allosteric sites on each subunit

One covalent site (Tyr 397)

TOP VIEW

Regulation is cumulative


Glutamate + NH4+ + ATP Glutamine + ADP + Pi

Glutamine Synthetase

(Biosynthesis - anabolic)

-Ketoglutarate

(Degradation – catabolic)

Take Home: Shutting down the enzyme favors using glutamate as an energy substrate.

Not shutting down the enzyme keeps the cell in a biosynthetic mode.


Adenylylated tyrosine residue

O

Enzyme

O

C

H

P

O

2

A

d

e

n

i

n

e

O

O

Tyr

O

H

O

H

Covalent Modification by Adenylylation

O

C

N

H

C

O

O

2

C

H

C

H

2

2

C

H

C

H

2

2

+

+

H

C

N

H

H

C

N

H

+

A

D

P

+

P

+

N

H

+

A

T

P

i

3

3

3

C

O

O

C

O

O


Allosteric Effectors

Each inhibits Glutamine Synthetase (Favors boosting cell energy

or shutting down a pathway requiring glutamine )

AMP

(Low energy state exists, oxidize -Kg)

CTP

(End product of pyrimidine synthesis)

Histidine

Glycine

Amino acids that are allosteric effectors

Tryptophan

Alanine

Glucosamine

(Glutamine sufficient for amino sugar synthesis)

(Glutamine sufficient for pyrimidine synthesis)

Carbamoyl-PO4


Covalent Regulation

Adenylylation of Tyrosine 397 on EACH of the 12 subunits

Adenyl = group attached

Adenylylation = group attached and process

For example:

An acyl group attached via an acylation reaction is

ACYLACYLATION

OR

ACYLYLATION

(PRONOUNCED ACIL-LIL-ATION)


Covalent Regulation of

Glutamine Synthetase

2 Transferases

Each puts on and takes off groups

AT (Adenylyltransferase - adenylylates GS)

UT (Uridylyltransferase - uridylylates PII)

One Regulatory Protein

PII

Two States

AT-PII

Adenylates

AT-PII-UMP

Deadenylylates


Rules of engagement
Rules of Engagement

  • Transferases catalyze adenylylation (uridylylation) and deadenylylation (deuridylylation reactions

  • Adenylylation shuts GS down cumulatively Deadenylylation turns GS back on cumulatively

  • AT requires PII to adenylylate

  • AT requires PII-UMP to deadenylylate


AMP

AT

AT

AT

AT

AT

AMP

GS

PPi

UTP

PII

PII

PII

PII

PII

PII

UMP

UMP

UMP

Uridylate removing

Enzyme

UR

UR

UT

UT

Active

GS*

ATP

Less Active

UMP

= Useless

= Uridylylates

= Adenylylates

(2 activities on

same enzyme)

= Deadenylylates

= Deuridylylates


Deadenylylates

UTP

PPi

UR

PII

PII

PII

PII

UMP

UMP

UR

UMP

UT

UT

Leads to adenylylation

Activates

GS

ATP

(-) glutamine

(+) -Kg

Inactivates

GS

Responsive to cell’s

nitrogen requirements


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