Amino metabolism - AA I

Question 1

Draw the structure of glutamine, glutmatate and α-ketoglutarate.

Answer 1

Question 2

Briefly describe the general pathway of ingested N.

Answer 2

food proteins ingested,
then reach intestinal lumen: are cleaved

1. by proteases to peptides
2. by peptidases to amino acids
3. by bacteria to NH₃

_​⇒ absorbed by liver

Question 3

In which forms is N excreted from the body?

Answer 3

• urea
• NH₄⁺
• also: creatine, urate

Question 4

When can nitrogen balance be considered positive, or negative?

Examples for each.

Answer 4

• negative: excreted amount of N > ingested amount of N
  → starvation, diseases, senescence
• positive: N_in > N_ex
  → children, pregnancy, refeeding

Question 5

What are the 2 functions of proteases?

In which form are they produced?

Answer 5

• digestion of exogenous proteins (food)
• degradation of endogenous proteins (protein turnover)

BUT: produced as zymogens
→ need proteolitic activation

Question 6

What are endopeptidases?

Examples.

Answer 6

break peptide bonds of nonterminal amino acids (i.e. within the molecule)

• pepsin, enteropeptidase
• Ser proteases (trypsin, chymotrypsin, elastase)

Question 7

What are exopeptidases?

Classify.

Answer 7

peptidase that catalyzes the cleavage of the terminal peptide bond

• carboxypeptidases
• aminopeptidases
• di-, tripeptidases

Question 8

Which enzymes facilitate protein turnover intra- and extracellularly?

Answer 8

• extracellular: collagenases, metalloproteases, ellastases
• intracellular: cathepsins (in lysosomes), ubiquination

Question 9

What is the fate of the amino group?

Answer 9

it is either

• transaminated → used for biosynthesis of AAs
• deaminated → NH₄⁺ formed
• incorporated in form of NH₃ into organic compounds (e.g. for excretion via urine)

Question 10

What is a transamination reaction?

Which group of enzymes catalyzes it?

Answer 10

catalyzed by transaminases

reversible exchange of an amino- and an oxo-group
→ 2 pairs required, 1 always: α-KG + glu

Question 11

What are 2 of the most important transaminases?

Which reactions are catalyzed by them?

Answer 11

ALAT = Ala aminotransferase
_{(clinically: GPT = glu-pyruvate transaminase)}

α-KG + Ala → Glu + pyruvate

ASAT = Asp aminotransferase
_{(clinically: GOT = glu-OXA transaminase)}

α-KG + Asp → Glu + OXA

Question 12

What is the clinical relevance of ALAT and ASAT?

Answer 12

non-functional plasma enzymes
= markes of tissue damage

• ALAT = liver (in cytosol)
• ASAT = heart/muscle (in mito.)

→ De Ritis ratio:

• ASAT/ALAT < 1 → minor liver damage
• ASAT/ALAT > 2 → severe mit. damage, myocardial infarcts, severe muscle damage

Question 13

What is the prostethic group of transaminases?
General function?

It is a derivate of which vitamin?

Structure(s).

Answer 13

pyridoxal-P = der. of vit B₆
bound to a Lys residue of enzyme as a Schiff base

⇒ transfers amino-group from AA to ketoacid
pyridoxal-P (PLP) → pyridoxamine-P (PMP)

_{Schiff base = aldehyde, but N instead of O}

Question 14

In which enzymes can PLP be found as the prostethic group?

Answer 14

• transaminases
• amino decarboxylases
• D/L amino racemases
• Ser-hydroxymethyltransferase
• Ser-Thr dehydratase
• cystathione synthase

Question 15

Describe the mechanism of transamination.

Answer 15

1. PLP + amino-group → aldimine + H₂O
2. forming further Schiff base tautomers
3. C-N double bound is cleaved → ketimine
4. ketimine + H20 → α-ketoacid + pyridoxalamine-P

• *​**then reverse reaction, amino-group transferred
• *to α-KG to form glu**, PLP reformed

Question 16

What is deamination?

List the 5 types.
Which one is the most important one?

Answer 16

amino group of AA is released as NH4+​

• oxidative deamination: loss of α-amino group (via GLDH)e
• O₂ dep. ox. deamination: loss of α-amino group w/ flavoproteins
• eliminating deamination: loss of α-amino group during catabolism
• hydrolytic deamination: loss of β-, μ-amino group
• deamination via nucleotide metabolism

Question 17

Which enzyme catalyzes oxidative deamination?

Where is this enzyme located in the cell?

Reaction + structures.

Answer 17

glutamate dehydrogenase = GLDH
mitochondrial enzyme, conversion of Glu to α-KG

Glu + NAD⁺ + H₂O → α-KG + NADH/H⁺ + NH₄⁺

or: reverse reaction can be used to incorporate free ammonium into org. compounds

_{via formation of intermediate, not relevant though}

Question 18

How is GLDH regulated?

Therefore, what is its main function?

Answer 18

regulated allosterically by

• ATP/GTP → inhibited, act. reverse reaction
• (cf. own card)*
• ADP/GDP → activated

⇒ dependent on energy status of cell, main function is to provide substrates for citrate cycle (α-KG, NADH/H⁺) rather than fixation of NH₄⁺

Question 19

Where can the 2 isoenzymes of GLDH be found?

Answer 19

glutamate dehydrogenase

• GLUD 1: in liver, kidney, other tissues
• GLUD 2: in brain, testis

Question 20

Which enzymes catalyze hydrolytic deamination?

What is it?

Reactions.

Answer 20

AA hydrolyzed to release NH₄⁺

• glutaminase: Gln + H₂O → Glu + NH₄⁺
• asparaginase: Asn + H₂O → Asp + NH₄⁺

Question 21

Which enzyme is responsible for eliminating deamination?

Reaction.

Answer 21

Ser-Thr dehydratase
uses PLP as prosthetic group

catalyzes deamination in 2 steps:

1. Ser/Thr + PLP → H₂O + aminoacrylate
2. aminoacrylate + H₂O → PLP + pyruvate (Ser)/α-ketobutyrate (Thr)

Question 22

Which enzymes catalyze O₂ dependent oxidative deamination reactions?

Differentiate.

Answer 22

AA oxidases
use O₂ to deaminate AA to α-ketoacids via electrontransfer to flavoproteins, stereospecific

• D-AA oxidases: in peroxisomes, use FAD
  → prod. H₂O₂ used for detoxification
• L-AA oxidases: in ER, use FMN
  → less important bc L-AA not physiological

Question 23

What is transdeamination?

Function.

Answer 23

process where a transamination is linked to a second reaction which results in deamination

→ action of transaminase, then GLDH

• glucose/energy provided via α-ketoacid
• energy provided via NADH
• NH₄⁺ excreted

Question 24

What is the function of the urea cycle?

Where does it happen?

Which enzymes catalyze which step?

Answer 24

ONLY in LIVER

production of urea from ammonia that was incorporated into Gln, Ala
→ excreted via urine

1. carbamoyl phosphate synthetase I
2. ornithine transcarbamoylase
3. arginosuccinate synthetase
4. arginosuccinate lyase
5. arginase

(to minor extent in kidney)

Question 25

Which enzyme catalyzes the first step of the urea cycle? Reaction + structures. Where does the reaction take place?

Answer 25

**_carbamoyl phosphate synthetase I_** (CPT I) in mitochondrial matrix bicarbonate + ammonium form carbamoyl-P, 2 ATPs used _{carbamoyl contains first of 2 NH2 groups of urea}

Question 26

Which enzyme catalyzes the second step of the urea cycle? Reaction + structures. Where does the reaction take place?

Answer 26

**_ornithine transcarbamoylase_** in mitochondrial matrix carbamoyl group transferred to ornitihine, citrulline formed

Question 27

What must happen with ornithine before it can be further processed in the urea cycle? Explain.

Answer 27

since first 2 steps located in mitochondrial matrix, but step 3-5 in cytosol → **ORNT****1** antiporter in inner mit. membrane imports citrulline, exports ornithine to cytosol

Question 28

Which enzyme catalyzes the third step of the urea cycle? Reaction + structures. Where does the reaction take place?

Answer 28

**_arginosuccinate synthetase_** in cytosol condensation of citrulline + Asp, form arginosuccinate, ATP → AMP (2 ATP equivalents) _{second of 2 NH2 groups of urea provided by Asp}

Question 29

Which enzyme catalyzes the fourth step of the urea cycle? Reaction + structures. Where does the reaction take place?

Answer 29

**_arginosuccinate lyase_** in cytosol arginosuccinate cleaved to fumarate + Arg

Question 30

Which enzyme catalyzes the fifth step of the urea cycle? Reaction + structures. Where does the reaction take place?

Answer 30

**_arginase_** in cytosol arginine hydrolyzed to urea and ornithine _{(ornithine then transported via ORNT1 into mitochondria)}

Question 31

How much ATP is needed for the synthesis of urea? In which reactions?

Answer 31

**4 ATP equivalents** * 2 ATP for CPT I * 2 ATP equ. (ATP → AMP) for arginosuccinate synthetase

Question 32

What is the function of N-acetyl Glu? How is it produced?

Answer 32

**necessary to activate CPS I** = rate limiting step of urea cycle _produced from Nac-Glu synthase_ acetyl-CoA + Glu → N-acetyl Glu

Question 33

Which substances regulate the activity of Nac-Glu synthase? Consequence?

Answer 33

**arginine** → hence: production of urea in hepatocytes regulated by AA level, rather than [NH₃]

Question 34

What happens with fumarate after being synthesized in the urea cycle?

Answer 34

**fumarate** enters citrate cycle, used for gluconeogenesis _{(already in postabsorptive phase)}

Question 35

Where do Asp and Orn as substrates in the urea cycle come from?

Answer 35

* **ornithine** synthesized from Glu/α-KG = anaplerotic reaction * **Asp** synthesized from OXA (from citrate cycle) by ASAT in mitochondria, then exp. into cytosol

Question 36

What happens in case of a [NH₄⁺] so it cannot be all used for the urea cycle? Reaction.

Answer 36

_used by **GLDH** to **aminate α-KG → Glu**_ α-KG + NADPH + NH₄⁺ → Glu + NADP⁺ + H₂O __NOTE:_ _{requires NAPDH; NADH formed in deamination reaction}

Question 37

How is the urea cycle regulated?

Answer 37

* **allost. act.** of CPS I by N-acetyl-Glu → presence of Glu, energy * **enzyme induction** during high protein diet, starvation * **intermediate concentration** - Arg: act. Nac-Glu synthetase - Orn: de novo synthesis *(cf. own card)*

Question 38

What is a hyperammonemia? How is it generally treated?

Answer 38

_= defect of urea cycle_ → **GLDH uses α-KG for removal of NH₃**, hence citrate cycle is depleted, low ATP level _treatment:_ * **limited intake** of protein, glucose (insulin) * **inhibition** of bact. NH₃ release in colon (lactulose) * **administration** of AA binding chemicals: benzoate (Gly), phenylacetate (Glu) * **replacing** missing intermediates

Question 39

Distinguish btw hyperammonemia type I and II w/r/t * underlying enzyme defect * symptoms * treatment

Answer 39

**_type I = CPS I deficiency_** * **​***symptoms:* mental retardation * *treatment:* Arg supplementation to stimulate Nac-glu synthetase **_type II = ornithine transcarbamoylase_** most common form of hyperammonemia * *symptoms:* X-chromosal inheritance, mental retardation, orotic aciduria _{orotic aciduria = carbamoyl not used for synth of citrulline, hence citrulline used in cytosol for synthesis of pyrimidine bases}

Question 40

Which further enzyme defects/deficiencies are associated with the urea cycle? Describe w/r/t * symptoms * treatment

Answer 40

**_arginosuccinate synthetase def._** * *symptoms:* citrullinemia * *treatment:* Arg supplementation **_arginosuccinate lyase def._** * *symptoms:* arginosuccinate excretion * *treatment:* Arg supplementation **_arginase def._** * *symptoms:* high Arg level, abnormal CNS development * *treatment:* Arg-free diet

Question 41

Describe how the intestines contribute to the synthesis of Arg?

Answer 41

**_form citrulline to be transported to kidney for Arg synthesis_** 1. 25% of urea synthesized in liver enters intestine, cleaved by **urease** of intestinal bacteria urea → NH₄⁺ + HCO₃^- 2. produce carbamoyl-P via CPS I, then used w/ ornithine to synthesize **citrulline**

Question 42

Which organ is responsible for formation of Arg? Explain. What are the functions of the synthesized Arg?

Answer 42

_2-4g/d (∽ daily dietary intake) synth. in **kidneys**_ **citrulline from int. → urea cycle → Arg** ⇒ then released into circulation for synthesis of **NO** and **polyamines** in extrahep. tissues ⇒ Arg = only semi-essential AA _{(bc in liver high arginase activity to enhance NH4+ excretion, Arg merely secreted into blood stream)}

Question 43

Which enzyme is responsible for the synthesis of Gln? Why is it important? Reaction.

Answer 43

**_Gln synthetase_** together with CPS I most imp. reaction of fix NH₄⁺ **Glu + ATP + NH₄⁺ → Gln + ADP + P_i** ⇒ Gln is 1 of the 2 transport forms of amino nitrogen in blood, acts as **amino group donor** _{NOTE: reverse reaction cat. by glutaminase}

Question 44

Which enzyme is responsible for the synthesis of Asn? Reaction.

Answer 44

**_Asn synthetase_** Gln + Asp + ATP → AMP + PP_i + Glu + Asn _{NOTE: synthesis of Asn is irreversible, can only be used for protein synthesis}

Question 45

What are the 2 transport forms for amino nitrogen?

Answer 45

**Gln + Ala** ⇒ enter * glutamine cycle * alanine cycle

Question 46

Describe the release of the 2 transporter AAs Ala and Gln from muscle into the blood stream.

Answer 46

* **Ala** = formed from pyruvate (glycolysis) by ALAT * **Gln** = formed from Glu by glutaminase _{(proteolysis in muscle → BCAAs → products = substrates of citrate cycle → α-KG transaminated to Glu)}

Question 47

Describe the glutamine cycle (not in brain).

Answer 47

1. **Gln released** from muscle due, adipose tissue and lung into blood stream 2. absorbedy by **liver**, excess amount enters urea cycle 3. remaining Gln + urea **secreted** **into blood stream** 4. both taken up by **kidney** → urea excreted → Gln converted, used for gluconeogenesis, NH₃ used to neutralize urine

Question 48

Describe the alanine cycle.

Answer 48

1. **Ala rel.** from working muscle, intestine (food) into blood stream 2. taken up by **liver**, glucose reformed 3. **glucose** released again into blood stream → analogous to Cori cycle (lactate)