Nitrogen Metabolism: Ammonia Assimilation

Ammonia assimilation

The incorporating of ammonia back into a metabolic form

Ammonia is ___ at high concentrations

toxic

what levels must ammonia be kept at

5-10 microM

free ammonia is able to cross membranes, which..

dissipates proton gradients

free ammonia depletes a-KG, which ....

inhibits TCA activity

What are the 4 pathways for assimilation

1. Glutamate Dehydrogenase
2. Glutamine Synthetase
3. Carbamoyl-phosphate synthetase I
4. not important

Glutamate Synthetase

main way to trap ammonia

glutamine serves as a major _______ donor in biosynthetic reactions

NItrogen

glutamine is the major _________ shuttle

inter-organ nitrogen

Glutamine synthetase is a __________ via _________ rxns

double displacement, SN2

glutamate is phosphorylated by ________ to give the _____________ intermediate

ATP, y-glutamyl-P

y-glutamyl-P is an essential intermediate

true

y-glutamyl-P reacts with ammonia (as NH3) to produce __________

glutamine

how do we have neutral ammonia at a physiological pH?

pH 7.2 < pKa 9.2 so it should be mostly in the acidic form (NH4+)
the ammonium ion deprotonation rxn is driven by the deltaG of ATP
ATP hydrolysis generates 50 kJ/mol of E
deltaGdeprotonation = + 10 kJ/mol
Theres a conformation change that occurs in the enzyme that allows for the deprotonation to occur

Glutamine synthetase rxn

glutamate ---> glutamyl-P ---> glutamine

keeping opposing enzymes in separate cell compartments prevents futile cycles. t/f? give an example.

True
glutamine synthetase and glutaminase

Glutamine synthetase is exclusively found in the ______

cytoplasm

Glutaminase is exclusively found in the __________

mitochondria

Carbamoyl-Phosphate Synthetase I

main rxn for ammonia assimilation in the MITOCHONDRIA

CPS I is a _________step rxn

multi

CPS has _________bound intermediates

enzyme

CPS I step one looks like a ________________ rxn

glutamine synthetase

CPS I step 1

bicarbonate is phosphorylated by ATP to give the carboxyl-P intermediate
Carboxyl-P reacts with ammonia to produce carbamate

CPS I step 2

kinase-type phosphorylation
rxn is very energy dependent

how much ATP does CPS-I rxn use?

2 ATP

Carbamoyl-Phosphate Synthetase II

glutamine-dependent biosynthetic enzyme
glutamine is being used as the Nitrogen source (instead of NH3)
glutamine is crucial for efficient biosynthesis of nitrogen-containing metabolites

CPS II alread "paid" to .....

deprotonate ammonium ion with glutamine synthetase, now benefits from having ammonia where it is needed while avoiding ammonia toxicity in other cells

CPS II rxn occurs in the _______

Cytosol

in CPS II, utilizing ____________ and the ______________ allows for free ammonia without the cost of deprotonating

glutamine, the aprotic transfer tunnel

the transfer tunnel contains how many active sites

2 active sites

CPS II process step 1

1. Glutamine-hydrolyzing subunit

CPS II process step 2

2. The transfer tunnel

CPS II process step 3

3. Biosynthetic subunit

Glutamine hydrolyzing subunit is initially filled with _______, which is displaced by _____________

water, glutamine

glutamine-hydrolyzing subunit hydrolyzes _____________ to release __________

glutamine, free ammonia

why doesnt water enter the transfer tunnel?

water in W5 form is too big

in the glutamine-hydrolyzing subunit a y-thioester is formed t/f?
this thioester acts as a _______ to block proton entry t/f?

True, LID, true

In the transfer tunnel ________ is moved unprotonated between active sites

NH3

In the biosynthetic subunit _________ reacts with the intermediate

NH3

Glutamine-dependent rxns will have a _________ Km for glutamine
(_______ affinity)

LOW Km
therefore, high affinity for glutamine

CPS I produces:

carbamoyl-P

CPS II produces:

carbamoyl-P

CPS I location:

mitochondria

CPS II location:

cytosol

CPS I substrate

NH3

CPS II substrate

glutamine

CPS I affinity for NH3

high affinity

CPS II affinity for NH3

NONE

CPS I affinity for glutamine

NONE

CPS II affinity for glutamine

HIGH

CPS I Pathway

Urea cycle

CPS II pathway

pyrimidine nucleotide biosynthesis

CPS I activators

N-Acetyl Glutamate