heme synthesis required in biochemistry.pdf

Biochemistry practical

1. Metabolism of heme

2. Describe any pathway under the following subheadings.
•Background/purpose in brief
•Site (organ specific/ subcellular)
•Preparatory steps (if any)
•Main steps of synthesis or degradation. Explain each step with necessary reactions.
Draw complete
cycle to summarize
•Regulation of key enzymes
•Energetics (related pathways)
•Biochemical and functional significance of the pathway or byproducts
•Clinical significance of the pathway

3. HEME - Structure
PYRROLE RING PORPHYRIN
I
I
I
II
I
IV
α
βγ
δ
1 2
3
4
56
7
8

4. HEME - Structure
•HEME IS A METALLOPORPHYRIN
•METAL + PORPHYRIN
•METAL - Iron
•Porphyrin – 4 pyrrole rings connected through Methenyl
bridges(-CH=)

5. Side chains of porphyrin
CH
2
COOH
Acetate, A
CH
CH
2
Vinyl, V
CH
3
Methyl, M
CH
2
CH
2
COOH
Propionate, P

6. Distribution of side chain
M
M
M
M
V
V
P
P
A
A
A
P
P
P
P
A
A
A
A
A
P
P
P
P
M
M
M
P
P
P
P
M
M
M
M
M
P
P
P
P
UROPORPHYRIN I
UROPORPHYRIN III
COPROPORPHYRIN
I
COPROPORPHYRIN III
PROTOPORPHYRIN III

7. •Most water soluble – Uroporphyrin
•Least water soluble – Protoporphyrin

8. Type I series – substituent groups arranged
symmetrically & alternately
A
P
A
P
A
A
P
P
Uroporphyri
n I
M
M
M
P
P
P
P
Coproporphyr
in I
A – acetyl (-CH2-COOH)
M – methyl (-CH3)
P – propionyl (-CH2-CH2-
COOH)
M

9. Type III series – substituent groups arranged
alternately & asymmetrically
A
A
A
A
M
M
M
M
P
P
P
P
P
P
P
P
Uroporphyrin
III
Coproporphyri
n III

10. VM
N
H
V
M
HN
PM
H
N
M
P
NH
CH
2
CH
2
H
2C
H
2C
Protoporphyrinogen

11. VM
N
H
V
M
HN
PM
H
N
M
P
NH
CH
CH
HC
HC
Protoporphyrin

12. FERROPROTOPORPHYRIN
VM
N
V
M
N
PM
N
M
P
N
CH
CH
HC
HC
HEME is a
F
e

13. HEME CONTAINING PROTEINS
1.HEMOGLOBIN-transport of O2 in blood
2.MYOGLOBIN-storage of O2 in muscle
3.CYTOCHROME C- ETC
4.CYTOCHROME p450-hydroxylation of xenobiotics
5.CATALASE- degradation of H2O2
6.TRYPHTOPHAN PYRROLASE-oxidation of tryptophan

14. HEME METABOLISM

15. HEME
SYNTHESIS
HEME
CATABOLISM
Heme metabolism

16. HEME - BIOSYNTHESIS
It can be synthesized by almost all tissues in the body
Erythrocyte precursors of bone marrow- normoblasts (not
in mature
erythrocytes) -85%
Liver – majority of reminder
Organelle – partly cytoplasmic & partly mitochondrial

17. Step 1: Synthesis of ALA
•This step occurs in the mitochondria
Succinyl CoA + Glycine δ-aminolevulinic acid
(ALA) ALAS
Pyridoxal
phosphate
ALAS - aminolevulinic acid synthase
TCA
cycle

18. ALA Synthase – Rate limiting enzyme in Heme
biosynthesis
Pyridoxal phosphate deficiency-Anemia

19. ❖Two isoforms
1)ALAS 1-is a house keeping gene and is expressed
in all cells.
2)ALAS 2-Erythroid form and expressed in fetal
liver and
adult bone marrow
Out of which ALAS1 is the key regulatory enzyme
ALA synthase

20. Step 2: Synthesis of porphobilinogen (PBG)
ALA + ALA
PORPHOBILINOGEN (PBG)
ALA
dehydratase
(Zn
2+
)
CYTOSOL

21. ALA dehydratase (porphobilinogen
synthase )
•Zinc containing enzyme
•Inhibited by lead
•ALA is elevated in lead poisoning
• Present in cytoplasm

22. Step III-Formation of UPG
A
P
A
P
A
PA
P
Four molecule of
PBG
Hydroxy
methylbilane
- 4
NH
3
(PBG
deaminase,
HMB synthase)

23. A
P
A
P
A
PA
P
Hydroxy
methylbilane
Step III-Formation of UPG
Spontaneo
us
cyclization
Uroporphyrinoge
n III
Synthase

24. Step III-Formation of UPG
Cytoplas
m
Uroporphyrinoge
n III
Synthase

25. Step IV-Synthesis of CPG
UPG
III
Coproporphyrinoge
n III
4
CO
2
UPG
decarboxylase
Cytoplas
m
CPG III enter Mitochondria through the transporter
ABC-B6

26. •UPG decarboxylase is also capable of converting UPG 1 to
CPG1

27. Step V-Synthesis of PPG
Protoporphyrinoge
n III
CPG III
CO
2
CPG
Oxidase
Mitochond
ria
V
V
O
2
NADP
NADPH + H
+
CPG Oxidase is specific for CPG III. So type1 protoporphyrin
does not
Occur in nature

28. Step VI-Generation of PP
PPG
III
Protoporphyr
in
PPG
oxidase
Methylene
bridges
Methenyl
bridges
4
H
Mitochond
ria

29. Step VII-Generation of Heme
F
e Fe
F
e
Ferrochelatas
e
(Heme
synthase)
Mitochond
ria
Protoporphyr
in
Hem
e

30. Step 1: Synthesis of ALA
Succinyl CoA + Glycine δ-aminolevulinic acid
(ALA) ALAS
Pyridoxal
phosphate
ALAS - aminolevulinic acid synthase
TCA
cycle

31. Step 2: Synthesis of porphobilinogen (PBG)
ALA + ALA
PORPHOBILINOGEN (PBG)
ALA
dehydratase
(Zn
2+
)
•ALA dehydratase is a zinc requiring enzyme
•Also known as porphobilinogen synthase
CYTOSOL

32. Step 3: Synthesis of HMB
Step 4: Synthesis of UBG III
4 PBG
HYDROXYMETHYLBILANE (HMB)
HMB Synthase
UROPORPHYRINOGEN
III (UPG III)
UPG III Synthase
Linear
Asymmetric pyrrole
CYTOSOL
4NH
3

33. Step 5: Synthesis of CPG III
HYDROXYMETHYLBILANE
(HMB)
HMB Synthase
UROPORPHYRINOGEN
III (UPG III)
UPG III Synthase
(Linear)
(Asymmetric
pyrrole)
COPROPORPHYRINOGEN
III (CPG III)
UPG
decarboxylase
2 ALA
PORPHOBILINOGEN (PBG)
ALA
dehydratase
CYTOSOL

34. CPG III transported into mitochondria
HYDROXYMETHYLBILANE
(HMB)
HMB Synthase
UROPORPHYRINOGEN
III (UPG III)
UPG III Synthase
(Linear)
(Asymmetric
pyrrole)
COPROPORPHYRINOGEN
III (CPG III)
UPG
decarboxylase
2 ALA
PORPHOBILINOGEN (PBG)
ALA
dehydratase
CYTOSOL

35. PROTOPORPHYRINOGEN IX (PPG)
CPG III
Coproporphyrinogen
oxidase
2CO
2
Step 6: Formation of colorless PPG IX

36. Step 7: Formation of protoporphyrin IX
(PP IX)
PROTOPORPHYRIN IX PPG IX
CPG III
CPG III
oxidase
CO
2
PPG IX
oxidase

37. Final step: Synthesis of HEME
PP IX PPG IX CPG III
CPG III
oxidase
CO
2
PPG IX
oxidase
FERROCHELATASE
Fe
2+
HE
ME

39. Regulation of heme synthesis

40. Hepatic ALA Synthase
• Regulated by repression mechanism
•(Heme – co-repressor)

41. Heme
No ALA Synthase
RNAP

42. Heme absent
Promoter Regulator structural gene
ALA synthase

43. Compartmentalization of
enzymes

44. Hepatic ALA Synthase
•Hematin – Allosteric inhibitor
The rate of transport of ALA to the cytoplasm is
influenced by hematin levels in cells
•High conc. of glucose – prevent induction of ALA
synthase(Hypoglycemia activates PGC1 which induces ALAS1
gene)
•Drugs like INH – decrease availability of PLP

45. Other regulatory factors
•Drugs like Barbiturates , griseofulvin – induce heme
synthesis
•Lead– decrease heme synthesis by inhibiting ALA
dehydratase and Ferrochelatase

46. ALA
Synthase
ALA
dehydratase
Ferrochelat
ase
Hemat
in
High
glucose
PL
P
IN
H
Barbitura
tes
Lea
d
Lea
d

47. ALA
Synthase
ALA
dehydratase
Ferrochelat
ase
Hemat
in
High
glucose
PL
P
IN
H
Barbitura
tes
Lea
d
Lea
d

48. Regulation in Erythroid cells
• Not induced by drugs
• Regulation mainly at the level of Ferrochelatase & PBG
deaminase rather than ALA synthase
•Erythroid form does not produce feedback inhibition by
heme

49. Porphyrins are colored and fluoresce
•Porphyrinogens are colorless
•Porphyrins are colored substances and fluoresce
•Du to the presence of conjugated double bond in the
pyrrole ring and methylene bridges
•A characteristic feature of porphyrins is a sharp
absorption spectrum near 400 nm – Soret band
•Porphyrins emit red fluorescence when illuminated by UV light

50. ALA
Synthase
ALA
dehydratase
PBG deaminase and UPG III
synthase
UPG
decarboxylase
CPG
Oxidase
PPG
Oxidase
Ferrochelat
ase