Biochemistry Lecture 15 Revised

Amino Acid Metabolism

Study of nitrogen handling, amino acid breakdown, and biosynthesis in cells

Forms of nitrogen in metabolism

Nitrogen exists as N₂, NH₄⁺, amino groups, and incorporated biomolecules

Nitrogen fixation definition

Process converting inert atmospheric N₂ into biologically usable NH₄⁺

Nitrogen fixation equation

N₂ + 6H⁺ + 6e⁻ → 2NH₃

Non-biological nitrogen fixation conditions

Requires ~400°C and high pressure (Haber process)

Biological nitrogen fixation conditions

Occurs at room temperature and atmospheric pressure

Nitrogenase complex

Enzyme system responsible for biological nitrogen fixation

Dinitrogenase reductase function

Transfers electrons one at a time to dinitrogenase using ATP

Dinitrogenase function

Uses electrons to reduce N₂ to NH₃

Electron source in nitrogen fixation

Ferredoxin provides electrons

Energy cost of nitrogen fixation

16 ATP required per N₂ reduced

Reason nitrogen fixation is expensive

Breaking N≡N triple bond requires high energy

Cellular adaptation to fixation cost

Cells evolved nitrogen salvage pathways

Free ammonia toxicity

NH₃/NH₄⁺ is highly toxic to cells

Nitrogen assimilation strategy

Ammonia is incorporated into glutamate and glutamine

Glutamate role

Central amino group donor via transamination

Glutamine role

Carrier of activated nitrogen (amide group)

Glutamine function in biosynthesis

Donates nitrogen for nucleotides and complex molecules

Glutamate and glutamine significance

Gateway molecules for biologically accessible nitrogen

Glutamine synthetase reaction

Glutamate + NH₄⁺ + ATP → Glutamine + ADP + Pi

Glutamine synthetase function

Controls entry of nitrogen into the cell

Regulation of glutamine synthetase

Allosterically inhibited by many nitrogen-containing compounds

Examples of GS inhibitors

Histidine, tryptophan, carbamoyl phosphate, CTP, AMP, glycine, alanine

Nature of GS inhibition

Additive feedback inhibition

Biochemical logic of GS regulation

Recycling nitrogen is more efficient than fixing new nitrogen

Transamination definition

Transfer of amino group between amino acids and α-keto acids

Purpose of transamination

Allows interconversion of amino acids and funnels nitrogen

Key transamination pairs

Pyruvate ↔ Alanine

Oxaloacetate ↔ Aspartate

α-Ketoglutarate ↔ Glutamate

Rule of symmetry in transamination

Knowing keto acid reveals corresponding amino acid

Aminotransferases

Enzymes that catalyze transamination

PLP (pyridoxal phosphate) role

Main cofactor in amino acid metabolism

PLP mechanism

Acts as electron sink to weaken C–N bond

PLP ↔ PMP conversion

Intermediate carrier of amino group

Type of reaction for PLP

Double displacement mechanism

Glutamine amidotransferase function

Transfers amide nitrogen from glutamine to substrates

Glutamine amidotransferase reaction

Glutamine + R-OH → Glutamate + R-NH₂

Purpose of glutamine amidotransferases

Provide nitrogen for biosynthesis

Protein breakdown result

Produces free amino acids

Fate of amino acids in catabolism

Nitrogen removed, carbon skeleton used for energy

Problem of nitrogen removal

Cells must prevent toxic ammonia accumulation

Nitrogen transport form in blood

Glutamine transports NH₄⁺ safely

Reason glutamine is used for transport

Neutral and non-toxic carrier

Glutamine transport process

Glu + NH₄⁺ → Gln (tissues), Gln → Glu + NH₄⁺ (liver)

Alanine transport role

Transports nitrogen from muscle to liver

Alanine cycle reaction

Pyruvate + Glutamate → Alanine + α-Ketoglutarate

Reason alanine used in muscle

High pyruvate from glycolysis

Liver role in nitrogen metabolism

Collects amino groups and releases NH₄⁺

Take-home nitrogen handling

Nitrogen is transported to liver and converted to ammonia

Major nitrogen excretion forms

Ammonia, urea, uric acid

Ammonia characteristics

Toxic, raises pH, used by aquatic animals

Urea characteristics

Neutral, requires water for excretion

Uric acid characteristics

Insoluble, conserves water

Urea cycle purpose

Removes excess nitrogen safely

Location of urea cycle

Liver only

Cellular location of urea cycle

Mitochondria and cytoplasm

Big picture of urea cycle

Maximizes nitrogen removal while minimizing carbon loss

Entry of nitrogen into urea cycle

NH₃ enters as carbamoyl phosphate

Carbamoyl phosphate formation

NH₄⁺ + HCO₃⁻ + 2 ATP → Carbamoyl phosphate

Rate-limiting enzyme of urea cycle

Carbamoyl phosphate synthetase I (CPS I)

Location of CPS I

Mitochondrial matrix

Function of CPS I

Activates bicarbonate and incorporates first nitrogen

Urea cycle steps overview

Ornithine → Citrulline → Argininosuccinate → Arginine → Urea

Second nitrogen source in urea cycle

Aspartate

Argininosuccinate formation

Citrulline + Aspartate → Argininosuccinate

Argininosuccinate breakdown

Produces arginine and fumarate

Final step of urea cycle

Arginine → Urea + Ornithine

Link between urea and TCA cycles

Argininosuccinate shunt connects them

Fumarate fate

Enters TCA cycle → malate → oxaloacetate

Purpose of linkage

Recovers carbon and produces NADH

Importance of carbon balance

TCA cycle must remain carbon neutral

Reason urea cycle depends on TCA

Requires aspartate and energy

Regulation of urea cycle

N-acetylglutamate (NAG) acts as activator

NAG role

Senses nitrogen levels and activates CPS I

Condition increasing urea cycle flux

High nitrogen and sufficient energy

Energetics of urea cycle

Costs 4 ATP equivalents

Energy recovery

Fumarate metabolism generates ~2.5 ATP

Net energy cost of urea cycle

~1.5 ATP

Consequence of blocking fumarate → malate

Disrupts urea cycle

Reason for disruption

Loss of oxaloacetate → no aspartate

Additional effect

Fumarate accumulation inhibits enzymes

Conclusion of TTYP question

Urea cycle depends on TCA for both carbon and energy

Carbon skeleton fate after deamination

Used for glucose or ketone bodies

Glucogenic amino acids

Produce glucose via oxaloacetate

Ketogenic amino acids

Produce ketone bodies (acetyl-CoA)

Importance of metabolic hubs

Amino acids feed into key intermediates

Examples of hubs

Pyruvate, α-ketoglutarate, succinyl-CoA, oxaloacetate

Cofactors in amino acid metabolism

PLP, Biotin, THF, AdoMet

PLP role

Transamination

Biotin role

CO₂ transfer (carboxylation)

THF role

One-carbon transfer (various oxidation states)

AdoMet (SAM) role

Methyl group transfer

SAM synthesis cost

Requires 3 ATP equivalents

THF limitation

Cannot donate methyl groups directly

SAM advantage

Activated methyl donor

3-carbon rule in amino acid catabolism

3C amino acids → pyruvate

β-carbon functional group rule

Removed to yield clean 3C skeleton

Key concept: follow nitrogen

Track nitrogen from entry → transport → disposal

Nitrogen entry into cell

Via glutamine synthetase