Sulfur Containing Amino Acids
•
2AA: MET & CYS.
•
MET is converted to CYS through trans-sulfuration pathway
•
Cystine: dimer of CYS
•
Homocysteine: not a primary AA
METHIONINE
•
S CONTAINING; ESSENTIAL; GLUCOGENIC
FUNCTIONS:
1. METHYL DONOR: AS S-ADENOSYL METHIONINE (SAM).
‘Methyl’ is labile ‘cos of High energy bond
TRANS-METHYLATION REACTIONS INVOLVING ‘SAM’:
•
ETHANOLAMINE TO CHOLINE
•
GUANIDO-ACETIC ACID (GLYCOCYAMINE)
TO CREATINE (METHYL-GUANIDO-ACETIC ACID)
•
N-ACETYL SEROTONINE TO MELATONINE
•
CARNOSINE TO ANSERINE
•
NOR-EPINEPHRINE TO EPINEPHRINE
•
EPINEPHRINE TO METANEPHRINE
•
NICOTINAMIDE TO N-METHYL-NICOTINAMIDE
•
SERINE TO CHOLINE
•
tRNA TO METHYL Trna
2. CYSTEINE FORMATION (TRANS-SULFURATION REACTION)
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FATE OF ATOMS OF MET
Active Methyl Cycle
(i) MET is an EAA
(ii) But mammals can synthesize MET from Homocysteine if available (can replace MET in diet).
Can transfer a Methyl group back to Homocysteine from
5 Methyl THF (Tetra-hydro-folate).
Therefore, regenerate MET.
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(iv) Crossroads for 2 important vitamins : Folic acid and Vitamin B12 (Vitamin B12 deficiency  Folate
deficiency (Folate trap)
(v) One of the major determinants whether Homocysteine  MET is level of 5 methyl THF.
SAM blocks 5 methyl THF production therefore, decreases MET production.
ATP
METHIONINE
PPi + Pi
S-ADENOSYL METHIONINE
METHIONINE
ADENOSYL TRANSFERASE
GSH
THF
ACCEPTOR
METHIONINE
SYNTHASE
METHYL TRANSFERASE
B12
METHYLATED
ACCEPTOR
METHYL
THF
ADENOSINE
HOMOCYSTEINE
H2O
ADENOSYL
HOMOCYSTEINASE
S-ADENOSYL HOMOCYSTEINE
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Serine
Homocysteine
Cystathionine beta
synthase
PLP
Cystathionine
Homoserine
Cystathionase
Cysteine
Alfa keto hydroxy
Butyrate
NH3
Glucose
H2O
CO2
TCA
Cycle
Propionyl Co A
T
R
A
N
S
S
U
L
F
U
R
A
TI
O
N
Succinyl Co A
Trans-Sulfuration Pathway
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Trans - Sulfuration pathway is analogous to transamination for AA
(i)
Major degradation pathway for MET in mammals
(ii)
End Product is CYS
(iii) Two RXNs, both use pyridoxal phosphate as a cofactor (as with transamination)
CYSTEINE
Non Essential (Synthesized from Serine + Methionine)
Glucogenic (via Pyruvate)
Present in large amounts in Keratin (Nails & Hair)
Degradation
1. Transamination:Cysteine is transaminated to Beta Mercapto Pyruvic acid  Pyruvic acid +
Sulfur
2. S may form Thiocyanate with CN/may be removed as H2S/ Elemental S/ Sulfite
3. On Decarboxylation, Cysteine forms Beta-Mercapto-Ethanolamine which is used in synthesis
of Co Enzyme A
Cysteine Importance:
(i) protein synthesis
(ii) catalytic site of enzymes where SH is a nucleophile
(iii) synthesis of compounds  CoA, glutathione, taurine
(iv) provides SO4 on complete oxidation
CYSTINE:
(i) Dimer of CYS
(ii) Formed through oxidation mediated by O2, Cu2+, Mn2+ or Fe2+
(iii) Little free in cells
(iv) Important in disulfide bond formation in intact proteins to provide bridges and stabilize
conformation of proteins.
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METABOLIC FATE OF CYSTEINE
PYRUVATE
CYSTEINE
CYSTINE
GLUTAMATE & GLYCINE
TAURINE
CO ENZYME A
BILE ACIDS
GLUTATHIONE
TAUROCHOLIC ACID
METABOLIC ROLE OF CYSTEINE
1. Formation of Taurine which
i.
participates in conjugation and excretion of Bile acids.(Taurocholate)
ii.
Modulates Calcium flux
iii.
Modulates Calcium binding
iv.
Is Inhibitory CNS neurotransmitter
Cysteine + O2 Cysteic acid Taurine + CO2
2. Maintaining the structure of Proteins by forming Disulfide bridges
3. With age there is decreased influx of Cys in cells & the deficiency of Cysteine is thought to
contribute to Aging
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4. Formation of Glutathione (Gamma glutamyl
cysteinyl glycine)
GLUTAMATE
CYSTEINE
ATP
GAMMA GLUTAMYL CYSTEINE
SYNTHETASE
ADP+Pi
γ-GLU-CYS
GLYCINE
ATP
GLUTATHIONE SYNTHETASE
ADP+Pi
GSH
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Glutathione plays a role in:
1. Amino acid absorption
2. Activation of many enzymes having SH groups.
These enzymes are active in reduced form.
3. Maintaining integrity of RBC membranes (by
scavenging free radicals formed inside RBC.
Deficiency of G6PD leads to decreased
NADPH production & so,decreased
regeneration of Reduced GSH: this leads to
Hemolysis
O2.- superoxide dismutase H2O2 glutathione peroxidase H2O
2GSH
Glucose 6 phosphate
2NADP+
GS-SG
NADPH+ H+
Glucose 6 phosphate Dehydrogenase
3. Conversion of Met-Hb (Fe3+) to Normal Hb (Fe2+)
2Met-Hb-Fe3+ + 2GSH  2Hb-Fe2+ + GS-SG
4. Conjugation for Detoxification
Glutathione transfers its Cysteinyl group to several
compounds & helps in their detoxification:
1. Organo-phosphorous compounds
2. Alkyl/ Aryl Halides
3. Nitrogeous substances (eg. Chloro dinitro benzene)
4. Heavy metals
5. Drugs
GSH + R-X Glutathione S transferase Glu-Cys-R-Gly + X-H
GGT
Glu-Cys-R-Gly +acceptor
Cys-R- Gly +
Glu- acceptor
Peptidase
Cys-R- Gly
Cys-R + Gly
Cys-R + Acetyl Co A
N-Acetyl-Cys-R(exc)
N Acetyl transferase
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METABOLISM OF SULFUR
Sulfur in body is Organic + Inorganic
Organic: In proteins (S-amino acids), Sulfatides, Glycosaminoglycans (GAGs).
Inorganic: Derived from S-amino acids during Trans-sulfuration/ De-sulfuration reactions.
•
H2S derived from Cysteine may be oxidezed to Sulfites and Thiosulfates.
•
Urinary Sulfur is in the form of Inorganic Sulfates, Organic/ Ethereal Sulfates & Neutral Sulfur.
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• Active Sulfate/ Phospho-Adenosyl-PhosphoSulfate (PAPS)
• 2 ATP + SO42-  PAPS + ADP +Ppi
O
HO-S
O
O
O
P
OH
ADENINE
O
RIBOSE
PO3
•
PAPS participates in Sulfuration Reactions eg.
Synthesis of Sulfatides/ GAGs etc.
•
HOMOCYSTEINE
Homocysteine is unstable & when in excess
converts to Homocystine (dimer)
NH2
NH2
S-CH2-CH2-CH
HS-CH2-CH2-CH
COOH
COOH
NH2
HS-CH2-CH2-CH
NH2
S-CH2-CH2-CH
COOH
•
COOH
Normal serum
Homocysteine is 5-15μmol/L
•
Normal urine Homocystine is too low to detect
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Homocysteine levels are increased (Homocystinemia & Homocystinuria) due to:
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1. Decreased conversion to Cystathionine
2. Decreased conversion back to Methionine
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Homocystinemia (& Homocystinuria) is a risk factor for Cardiovascular disease
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Homocystine normal value: 5-15 micromoles/ L
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Raised (upto 100 times) in :
1. Aged,
2. Males
3. Post menopausal females
4.
Smokers,
5. Alcoholics,
6. Hypothyroidism,
7. CRF (kidney can’t excrete homocystine)
8. B6/ B12/Folic acid Deficiency,
9. Drugs that are Folic acid antagonists/ B6 antagonists/ B12 antagonists/ Estrogen antagonists/
Nitric oxide antagonists,
10. Congenital enzyme defects
High Serum Homocystine causes:
Increased risk of Atherosclerosis, CAD, Thrombo-embolism, Mental retardation
Prevention: B6, B12, Folic acid intake
Homocysteine interacts with Lysyl residues of Collagen Interfers with Collagen crosslinking.
Forms Homocysteine Thiolactone (free radical) thiolates LDL these LDL endocytosed by
Macrophages Atherosclerosis.
High maternal Homocysteine marker for fetal Neural tube defect.
Prevention: Folic acid in 1st trimester of pregnancy
Excess Homocystine can spill over to urine: Homocystinuria.
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Homocystinurias
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(2nd most common genetic AA disease)
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Autosomal recessive, 1 in 2 lakh live births
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 Homocysteine in blood.
•
Homocystinuria.
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4 types:
Type I: Defect in cystathionine beta synthase
(converts Homocysteine  Cystathionine)
•
Increased Methionine and Homocystine in urine
•
Low serum Cysteine
•
Signs: Mental retardation, Charlie chaplin gait, Skeletal deformities, Ectopia lentis, Myopia,
Glaucoma, Increased platelet adhesiveness, Intravascular thrombosis
•
Treatment: “Low Methionine & High Cysteine” diet,PLP
•
Type II: Defect in methylene THF reductase
•
(converts Methylene THF to methyl THF)
•
So, Decreased [5 methyl THF] Decreased Methionine
•
Homocystinuria
•
Behavioral changes, Vascular abnormality.
•
Treatment: Folic acid
•
Type III: B12 deficiency
•
So, decreased Methionine formation
•
Low serum Methionine
•
High serum Homocysteine
•
Homocystinuria
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Type IV: Malabsorption of B12 from gut
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So, decreased Methionine formation
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Low serum Methionine
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High serum Homocysteine
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Homocystinuria
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Cystinuria (Cystine-Lysinuria)
•
Most common inborn error of amino acid transport
•
Autosomal recessive.
•
Normally these a acids are reabsorbed in PCT by Specific carriers ( 1 for Cystine, 1 for Lysine,
Ornithine, Arginne, 1 for all 4 (COLA)
•
Cystinuria is caused by defect of 3rd type of Carrier protein.
Leads to:
1. Excess urine excretion of Cystine, Ornithine, Lysine and Arginine.
2. Crystalluria (Cystine crystals in acidic urine): Cystine is least soluble of all natural a. acids:
so,forms Cystine stones in Renal pelvis, Ureter or Bladder
3. leading to Obstructive uropathy
Treated by increasing solubility of Cystine: more fluids, urine alkalizers (NaHCO3), Reducing
Cystine excretion using D-penicillamine/ Captopril
Cystinosis
Autosomal recessive
Abnormality in Cysteine transport
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Cystine crystals deposit in Lysosomes in Liver, Spleen, Bone marrow, WBC, Kidneys, Cornea,
Lymph nodes
Treatment as in Cystinuria + D-Penicillamine
Hyper Methininemias
Due to: 1. Impaired utilization
2. Excess formation
3. Liver disease
Oasthouse syndrome
Defective Methionine absorption
Excess urine excretion of Methionine, Aromatic and Branched chain amino acids.
Homocystinuria Case Discussion
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A 6-year-old girl was brought to the hospital with vision problems.
•
She was found to have a downward dislocation of the left lens.
•
Her mother indicated that the girl’s birth was normal, but that she lagged in development.
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She was unable to crawl until 1-year-old and did not walk until 2 years.
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Speaking was also delayed.
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She had long, thin bones; on X-ray the lower femur showed signs of osteoporosis.
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An older brother had similar symptoms and had been diagnosed as having Marfan’s
syndrome.
•
A simple cyanide-nitroprusside test of the patient’s urine was positive, suggesting
homocystinuria.
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•
This was confirmed by amino acid analysis of the plasma, which revealed Homocysteine, an
abnormally high methionine level, and other sulfur-containing compounds that were
derivatives of homocysteine.
•
The patient was treated with a low-methionine diet supplemented with folic acid and
pyridoxine.
Symptoms / clinical characteristics
1. Downward dislocation left lens
Slow development - crawling / walking / speaking.
Osteoporosis, long thin bones
2. Cyanide/nitro prusside urine test +ve
3. Plasma AA   homoCYS, MET, homoCYS derivatives
Suggests that homoCYS  due to decrease conversion to CYS. MET also  ( therefore no problem
in conversion homoCYS MET)
QUESTIONS
1. What is the origin of the Homocysteine excreted in this disease?
2. What are some of the metabolic substances formed by the enzymatic reactions that use Sadenosylmethionine as the methylating agent?
3. What are some causes of homocystinuria in humans?
4. How would one test for a deficiency of cystationine βsynthase in this patient?
5. Explain why pyridoxine is useful in the treatment of some patients with homocystinuria.
6. What effect would a diet low in folate have on this patient?
7. What might account for the homocystinuria of an apparently normal infant (not in this case)
with severe megaloblastic anemia and who was exclusively breast-fed by a strict vegetarian
mother?
8. Describe the genetics of homocystinuria (cystathionine β synthase deficiency).
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ANSWERS
1. Origin of Homocysteine: dimer of homoCYS, product of excess homoCYS, overflows into urine
2.These include RNA, DNA, amino terminal groups P’s, precursors of melatonin, creatine,
epinephrine, phosphatidyl choline, methyl cobalamin
3. Any part of pathway that impedes MET  homoCYS pathway  homoCYS
- decreased cystathionine B synthase (Step 1 for CYS)
- decreased syn of MET from homoCYS (5 MeTHF or B12 or enzyme ) genetic or nutritional
- Also 6 azauridine administration: anticancer agent which inhibits PPal enzymes, therefore
decreases both step 1 and 2
- bacterial action on cystathionine in urine
4. Test of enzyme: Measure enzyme in cells (human skin fibroblasts)to define hetero or complete
mutation. Test addition of pyridoxal phosphate in vitro.( B6 sensitivity)
5. (i) pyridoxine and/or B6  improvement in behaviour and IQ
(ii) Diet decreased MET, add smaller meals to prevent MET overload
(iii) add Vitamin B12 and folic acid
6. Decreased folate in Diet : Good or Bad? BAD Seriously effect 5 MeTHF rxn therefore 
homoCYS even worse (no alternate pathway)
7. HomoCYS  B12 deficiency
- vegetarians: no eggs, no dairy, scrubbed vegetables  decrease cobalamin therefore not
enough vitamin B12  synthesize cofactor, therefore no homoCYS  MET
Can result in permanent neurological damage
8. Autosomal Recessive (1:45,000), Defects in gene chromosome 21
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