biochemistry of role of cysteine as mentioned in dr
TRANSCRIPT
Biochemistry of Role of cysteine as mentioned in Dr
Douglin’s lecture
• Role of the NADH generated in the PPP for Superoxide detoxif ication & the pathophysiology of hemolytic crises
PENTOSE PHOSPHATE PATHWAY ASSISTS GLUTATHIONE PEROXIDASE IN PROTECTING ERYTHROCYTES AGAINST HEMOLYSIS
• Besides generating reducing power in the cytosol in the form of NADPH for anabolic purposes such as the biosynthesis of fatty acids, steroids and related compounds, the PPP also
• generates NADPH for protection against free radicals in erythrocytes (Superoxide detoxif ication) and for other metabolic purposes, (like producing free radicals in neutrophils to kill bacteria!)
• 1. Free radical species can form in RBC’s and other tissues.
• These reactive free radical species and damage cell membranes, and cause hemolysis in RBC’s
Pentose phosphate pathway protects cells against reactive oxygen species (ROS)
• Molecular oxygen and partially reduced, reactive forms of oxygen may be produced.
• Reduction of molecular O2 in a series of one-electron steps yields superoxide, hydrogen peroxide, hydroxyl radical, and water.
• These intermediate, activated forms of oxygen are known as reactive oxygen species (ROS)
• Reactive Oxygen Species
• When molecular oxygen (O2) is partially reduced, unstable products called reactive oxygen species (ROS) are formed.
• These react rapidly with lipids to cause peroxidation, with proteins, and with other substrates, resulting in denaturation and precipitation in tissues.
• Reactive oxygen species include:
• Superoxide (O2.-)
• Hydrogen peroxide (H2O2)
• Hydroxyl radical (OH')
• The polymorphonuclear neutrophil produces these substances to kill bacteria in the protective space of the phagolysosome during the oxidative burst accompanying phagocytosis.
• Production of these same ROS can occur at a slower rate wherever there is oxygen in high concentration.
• Small quantities of ROS are inevitable by-products of the electron transport chain in mitochondria.
• These small quantities are normally destroyed by protective enzymes such as catalase.
• The rate of ROS production can increase dramatically under certain conditions, such as reperfusion injury in a tissue that has been temporarily deprived of oxygen.
• ATP levels will be low and NADH levels high in a tissue deprived of oxygen (as in an MI). When oxygen is suddenly introduced, there is a burst of activity in the ETC, generating incompletely reduced ROS.
• Defenses against ROS accumulation are particularly important in highly aerobic tissues and include superoxide dismutase and catalase.
• In the special case of erythrocytes, large amounts of superoxide are generated by the spontaneous dissociation of the oxygen from hemoglobin (occurrence is 0.5-3% of the total hemoglobin per day).
• The products are methemoglobin and superoxide. The processes that adequately detoxify the superoxide require a variety of enzymes and compounds, including superoxide dismutase, catalase, as well as glutathione peroxidase, vitamin E in membranes, and vitamin C in the cytoplasm.
• Low levels of any of these detoxifying substances result in hemolysis. For example, inadequate production of NADPH in glucose 6-phosphate dehydrogenase def iciency results in accumulation of the destructive hydrogen peroxide (
Role of NADPH in the RBC• Production of superoxide
– Hb-Fe2+-O2 -> Hb-Fe3+ + O2-.• Spontaneous rxn, 1% per hour
• O2-. + 2H2O -> 2H2O2• Both O2-. & H2O2 can produce reactive
free radical species, damage cell membranes, and cause hemolysis as indicated in the next two slides.
Oxidizing agent:
H2O2
OH
O2
Oxidative damage
2H2O
2G-SH G-S-S-G
NADP+ NADPH.H+
Pentose Phosphate Shunt
Glutathione peroxidase
Glutathione reductase
Mitochondrial respirationIonizing radiationSulfa drugsHerbicidesAntimalarial drugs
Hydroperoxyll
Superoxide
Detoxif ication of Superoxide Anion and Hydrogen Peroxide requires
• Antioxidant enzymes like– Superoxide dismutase
– Glutathione peroxidase
– Glutathione reductase
What is glutathione ?and What does it do? Glutathione is a tripeptide that includes a Glu linked by an isopeptide bond involving the side-chain carbonyl group. Its functional group is a cysteine thiol. One role of glutathione is degradation of hydroperoxides, that arise spontaneously in the oxygen-rich environment in red blood cells as described above. Hydroperoxides can react with double bonds in fatty acids of membrane lipids, making membranes leaky.
H3N+
HC CH2 CH2
COO
C
O
NH
CH
CH2
SH
C
O
NH
CH2 COO
-glutamyl-cysteinyl-glycine Glutathione
Glutathione Peroxidase catalyzes degradation of organic hydroperoxides by reduction, as two glutathione molecules (represented as GSH) are oxidized to a disulf ide.
2 GSH + ROOH GSSG + ROH + H2OGlutathione Peroxidase uses the trace element selenium as functional group. The enzyme's primary structure includes an analog of cysteine, selenocysteine, with Se replacing S.
H3N+
HC CH2 CH2
COO
C
O
NH
CH
CH2
SH
C
O
NH
CH2 COO
-glutamyl-cysteinyl-glycine Glutathione
Glutathione keeps proteins with reduced sulfhydryls
SH from oxidizing to R S S R’
P-SH + P’-SH + O2 P-S-S-P’ + H2O
P-S-S-P’
G-SH
P-SH + G-S-S-P
G-SH
G-S-S-G + HS-P
Glutathione reductase contains FAD
Reaction of glutathione with peroxides
2GSH + RA-O-O-H G-S-S-H + ROH +H2O
A steady supply of glutathione is required forerythrocyte integrity
~ 400,000,000 individuals are deficient in glucosedehydrogenase!
Without a fully functioning glucose dehydrogenase,glutathione concentrations Hemolytic Anemia can
occur if certain drugs are used.
• As indicated, reduced glutathione (GSH) protects the cell by destroying hydrogen peroxide and hydroxyl free radicals.
• But once reduced, glutathione (GSH) must be regenerated!
•
…..because reduced glutathione is needed forglutathione peroxidase, which destroy hydrogenperoxide and organic peroxides. This enzymerequires selenium as a cofactor.
2
H3+N CH
COO-
CH2 CH2 C NH
O
CH
CH2
C
O
NH CH2 COO-
H3+N CH
COO-
CH2 CH2 C NH
O
CH
CH2
C
O
NH CH2 COO-
S
SH3+N CH
COO-
CH2 CH2 C NH
O
CH
CH2
C
O
NH CH2 COO-
SH
• Regeneration of reduced glutathione (GSH) from it’s oxidized form (GS-SG) requires the NADPH produced within erythrocytes in the the glucose 6-phosphate dehydrogenase reaction in the Pentose Phosphate Pathway.
Glutathione Reductase catalyzes:
GSSG + NADPH + H+ 2 GSH + NADP+ Genetic def iciency of Glucose-6-P Dehydrogenase can lead to hemolytic anemia, due to inadequate [NADPH] within red blood cells.
The effect of partial def iciency of Glucose-6-phosphate Dehydrogenase is exacerbated by substances that lead to increased production of peroxides (e.g., the antimalarial primaquine).
• THE PENTOSE PHOSPHATE PATHWAY ASSISTS GLUTATHIONE PEROXIDASE IN PROTECTING ERYTHROCYTES AGAINST HEMOLYSIS thus
• The pentose phosphate pathway in the erythrocyte provides NADPH for the reduction of oxidized glutathione to reduced glutathione catalyzed by glutathione reductase, a f lavoprotein enzyme containing FAD.
• In turn, reduced glutathione removes H202 from the erythrocyte in a reaction catalyzed by glutathione peroxidase, an enzyme that contains the trace element selenium. This reaction is important, since accumulation of H202 may decrease the life span of the erythrocyte by increasing the rate of oxidation of hemoglobin to methemoglobin.
• REVIEW• NADPH is generated by the pentose phosphate
pathway.• One of the uses of NADPH is in the reduction of
oxidized glutathione to reduced glutathione.• Glutathione is necessary for the removal of H2O2
and lipid peroxides generated by reactive oxygenspecies.
• In the erythrocyte of healthy individuals, thecontinuous generation of superoxide ion from thenonenzymatic oxidation of hemoglobin provides asource of reactive oxygen species.
• Since these cause disease we can see howimportant the PPP is in producing NADPH.
• REMEMBER that the functions of NADPH generated in the PPP are that
• It is required by the cells for the following functions
• biosynthesis• maintenance of supply of reduced
glutathione to act as an antioxidant protect against reactive oxygen species “ROS” in RBC’s
• Generation of superoxide for the bactericidal activity of polymorphonuclear leukocytes (PMN- neutrophils)
• Detoxif ication (Cytochrome P-450 System) – liver