N metabolism and Urea Cycle Flashcards
what are aa’s catabolized to?
urea + CO2 + H2O
aa’s are used in the biosynthesis of what special molecules?
- porphyrins
- purines and pyrimidines
- NO
- melanin
- hormones, neurotransmitters
- creatine
zymogens
inactive enzymes
what does the stomach secrete for protein digestion?
- HCl
- pepsinogen
what is HCl secreted by?
parietal cells
what is low HCl called and what does it cause?
achlorhydria - iron deficiency
what is pepsinogen secreted by?
chief cells
how is pepsinogen turned into pepsin?
autocatalytic activation by conformational change at low pH
action of active pepsin
endopeptidase - cleaving peptide bonds to produce smaller peptides and some free aa’s
-prefers CO group provided by an aromatic or acidic aa
what enzymes does the pancreas secrete for protein digestion? how are they activated?
zymogens activated by proteolysis:
- trypsinogen -> trypsin
- chymotrypsinogen -> chymotrypsin
- proelastase -> elastase
- procarboxypeptidases -> carboxypeptidase A or B
what converts trypsinogen -> trypsin?
enteropeptidase
what converts chymotrypsinogen -> chymotrypsin?
trypsin
what converts proelastase -> elastase?
trypsin
what converts procarboxypeptidases -> carboxypeptidase A and B?
trypsin
pancreatic enzymes: which have endopeptidase activity and which have exopeptidase activity?
endo: trypsin, chymotrypsin, elastase
exo: carboxypeptidase A and B
where does trypsin cut?
after Arg, Lys (positive)
where does chymotrypsin cut?
after Trp, Tyr, Phe, Met, Leu (aromatics + randoms)
where does elastase cut?
after Ala, Gly, Ser
where does carboxypeptidase A cut?
before Ala, Ile, Leu, Val
where does carboxypeptidase B cut?
before Arg, Lys
how are aa’s absorbed?
semispecific Na+ transport proteins w/ at least 6 types of carriers (aa’s brought into cell with Na+ - symport)
what do defective transporters in aa absorption result in?
malabsorption from intestine and decreased resorption from glomerular filtrate -> Hartnup disease, cystinuria
what disease is caused by defective neutral aa transporters?
Hartnup disease
what is the mutation in Hartnup disease?
mutation in Na-dependent and Cl-independent neutral aa transporter, which is expressed predominantly in the intestine and kidneys
how is Hartnup disease passed down?
autosomal recessive
symptoms of Hartnup disease
- ataxia
- emotional instability
- pellagra rash (due to B3)
- neutral amidoaciduria
- high urine levels of neutral aa
what are most symptoms of Hartnup believed to be caused by?
niacin deficiency - need tryptophan to synthesize niacin, and with Hartnups, you can’t absorb tryptophan
what disease is caused by defective dibasic aa transporters?
cystinuria - also transport cystine
what is cystinuria?
cystine stones in bladder and kidneys
describe intracellular proteolysis of body proteins
- lysosomes: acid hydrolases, pH 5 optimum
- cytosol: proteins tagged with ubiquitin for degradation by 26S proteasome
describe extracellular proteolysis of body proteins
- serine proteases
- matrix metalloproteases (collagenases)
what can elastase from neutrophils contribute to clinically?
lung damage in chronic obstructive pulmonary disease
what can matrix metalloproteases contribute to clinically?
metastasis of cancer
how does the body deal with N formed from aa breakdown?
- blood: travels as aa’s (Ala, Glu)
- liver: efficiently detoxifies NH4, forms urea
what is the only organ that has all enzymes for urea synthesis?
liver
why does the body form urea?
major way body gets rid of ammonia
- soluble
- not charged (doesn’t change pH of fluids)
structure of urea
H2N -C(=O) -NH2
equilibrium for ammonia in body and pK value
NH3 + H+ NH4 pk = 9.3
problem with ammonia in body
ammonia (in equilibrium w/ NH4+) is very toxic to CNS
how is the problem with ammonia dealt with?
ammonia + ammonium converted to two nontoxic compounds: urea and glutamine
what is the first step in aa catabolism?
remove amino groups
two ways to remove the amino group from an aa?
- transamination
2. deamination
transamination
aminotransferase/transaminase moves alpha amino groups to an alpha-ketoacid to form glutamate, which is either used or deaminated, releasing an NH4+ (reversible rxn)
deamination
direct release of NH4+ - different methods for different aa’s:
- glutamate DH
- hydrolytic deamination (e.g. glutaminase)
- non-oxidative (PLP dependent)
what cofactor does transamination require?
PLP (pyridoxal phosphate)
reaction catalyzed by aspartate aminotransferases (AST)
Asp + alpha-ketoglutarate -> OAA + Glu
reaction catalyzed by alanine aminotransferase (ALT)
Ala + alpha-ketoglutarate -> pyruvate + Glu
why are AST and ALT important clinically?
serum levels used to evaluate for liver damage (especially for industrial solvents - chloroform, carbon tetrachloride)
where is PLP derived from?
vitamin B6
what reactions most commonly are associated with using PLP?
transaminations and deaminations - but also used for many other reactions (like glycogen phosphorylase)
business end of PLP?
aldehyde carbon forms a Schiff base (C=N)
three methods of deamination
- oxidative deamination
- hydrolytic deamination
- non-oxidative deamination (PLP dependent)
oxidative deamination
Glu DH: glutamate -> alpha-ketoglutarate + H+
- forms NAD(P)H from NAD(P), releases NH4+
- reversible
where does Glu DH rxn take place?
liver and mitochondria
inhibition of Glu DH
GTP, ATP - allosteric
activation of Glu DH
GDP, ADP - low energy increases aa breakdown to provide alpha-ketoglutarate for TCA
hydrolytic deamination
done w/ glutaminase or asparaginase on the side chain amide groups of glutamine and asparagine, respectively
glutaminase rxn
Gln + H2O -> Glu + NH4+
where is glutaminase important in the body?
kidney - NH4+ forms salts w/ metabolic acids for secretion
asparaginase rxn
Asn + H2O -> Asp + NH4+
non-oxidative deamination
done on serine or threonine by serine dehydratase and threonine dehydratase, respectively
what does non-oxidative deamination require?
PLP
serine dehydratase rxn
serine -> pyruvate + NH4+
threonine dehydratase rxn
threonine -> alpha-ketobutyrate + NH4+
in non-oxidative deamination, what precedes deamination?
dehydration
what aa’s transport nitrogen w/i body and where do they take it?
Ala and Gln - take N from other tissues to liver or kidney
Gln synthetase fxn
can fix NH4+ in a carbon compound - one of three human enzymes that can do this
what are mutations in Gln synthetase associated with clinically?
found in newborns w/ severe brain malformations - large amounts of glutamate in brain (Glu = neurotoxin)
pathway of ammonia transport to liver using Gln
a-KG -> Glu -> Gln in muscle/peripheral tissues ->
transported to liver -> Gln -> Glu -> a-KG, while NH4+ goes to make urea
N balance
excreted N = consumed N
-protein content exceeds requirement in normal healthy adult
when will you see a positive N balance?
- growing kid
- pregnant mom
- during physical trauma
- -when need to increase protein synthesis, N out < N in
when will you see a negative N balance?
- protein malnutrition
- starvation
- -body protein degraded to synthesize essential proteins, N out > N in
levels of NH4+ in blood
25-40 uM normally
overall rxn for urea cycle
NH4+ + CO2 + Asp + 3ATP + 2H2O –> urea + fumarate + 2ADP + AMP + PPi + 2Pi
how many high energy bonds are used for each molecule of urea made?
4 - 3 from ATP and one from PPi –> makes the rxn irreversible (large, negative deltaG)
origins of each of the parts of urea
- one NH2: from ammonia
- other NH2: from Asp
- carbonyl: from HCO3-
alternative pathways for forming urea
there are none - urea cycle is it
what can defects in urea cycle lead to?
an increase in NH4+ in blood -> hyperammonemia
where does the urea cycle take place?
some in cytosol and and some in mitochondria
CPS1 product
carbamoyl phosphate
urea cycle: general pathway
ornithine + carbamoyl phosphate -> citrulline -> (+ Asp) -> argininosuccinate -> (release fumarate) -> Arg -> urea + ornithine
where does CPS1 function?
in mitochondria
what cofactor does CPS1 require?
NAG (N-acetylglutamate) - allosteric activator
what is the rate limiting step for urea cycle?
CPS1 (carbamoyl phosphate synthase 1)
CPS1 vs. CPS2
CPS1: in mitochondria for urea synthesis
CPS2: in cytosol for pyrimidine synthesis
where is NAG synthesized?
in mitochondria by N-acetylglutamate synthase
Glu + acetyl coA -> NAG
NAG synthesis activators
Arg
which is catalytically active: monomer or dimer of CPS1?
monomer of CPS1-NAG
what enzyme: citrulline + Asp -> argininosuccinate
argininosuccinate synthetase
where does the argininosuccinate synthetase rxn take place?
cytosol
what enzyme: argininosuccinate -> fumarate + arginine
argininosuccinate lyase
what enzyme: arginine + H2O -> urea + ornithine
arginase
what enzyme: ornithine + carbamoyl -P -> citrulline
ornithine transcarbamoylase (OTC)
which urea cycle rxns occur in mitochondria?
- CPS1
- OTC
which urea cycle rxns occur in cytosol?
- argininosuccinate synthetase
- argininosuccinate lyase
- arginase
what does OTC deficiency cause?
increased orotate -> carbamoyl-P can’t react w/ OTC , so instead forms pyrimidines
what is diagnostic for OTC deficiency?
increase in pyrimidines diagnostic for OTC deficiency
short term regulation of urea cycle
depends on CPS1
(+) NAG - allosteric
(+) protein-rich meal - make more NAG (Km of NAG synthase for Glu is high - not saturated under physiological conditions
long term regulation of urea cycle
dietary protein levels regulate synthesis of urea cycle enzymes
- increase protein for days -> increase urea enzyme synthesis -> increase urea excretion
- reverse for low protein diet
starvation and the urea cycle
-starvation: increase protein degradation in tissues -> increase synthesis of urea cycle enzymes
links b/w urea cycle and TCA
- fumarate
- OAA:
- -transamination to Asp
- -conversion to glucose
- -condensation w/ acetyl coA to form citrate
- -conversion into pyruvate
what diseases can be caused by autosomal recessive inherited defects of urea cycle?
- HA type I (hyperammonemia)
- HA type II
- citrullinemia
- arginosuccinic aciduria
- argininemia
defect in HA type I and accumulations?
CPS1 - NH4+, Gln, Ala
defect in HA type II and accumulations?
OTC - NH4+, Gln, orotic acid
defect in citrullinemia and accumulations?
argininosuccinate synthetase - serum citrulline
defect in arginosuccinic aciduria and accumulations?
argininosuccinate lyase - arginosuccinate
defect in argininemia and accumulations?
arginase - arginine
mechanism of NH4+ toxicity
shift in equilibrium of Glu DH rxn toward direction of Glu formation
- could deplete a-KG (essential for TCA) -> decreased oxidation and ATP production
- brain: vulnerable to HA since depends on TCA for energy
what happens with high levels of Gln in brain?
- osmotic increase in H2O in astrocytes = brain swelling
- increased Gln causes decrease in neurotransmitters Glu and GABA
treatment for acute HA
hemodialysis or exchange transfusion to rapidly lower ammonia
treatment for defects in urea cycle
- limit protein intake in diet
- remove excess ammonia
- replace missing intermediates - supplement w/ Arg or citrulline, depending on where defect is
- future: liver cell supplementation?
how do you remove excess ammonia?
- Levulose
- avoid/treat infections (cell death releases proteins, aa’s)
- kill intestinal ammonia producing bacteria w/ antibiotics
- Benzoate and Phenylbutyrate
Levulose fxn
fructose - bacteria acidifies colon
-promotes excretion of ammonia
Benzoate and Phenylbutyrate fxn
reduce Gly and Gln levels
what drug did Phenylbutyrate replace?
Phenylacetate
4 ways to circumvent urea cycle defects in N excretion
- amino groups removed by scavenging aa’s for excretion
- benzoic acid removes Gly
- forces additional NH3 incorporation into aa synthesis
- phenylbutyrate removes Gln (has 2NH3 molecules each)
OTC deficiency
- X-linked
- usually presents in male children/young adults
- acts like dominant trait in het females - can get sick/die from HA
- male infants: explosive neonatal HA due to abnormal gene
- Jesse Gelsinger - first public gene therapy death
