introduction to metabolism. metabolism (the acquisition and utilization of free energy) catabolism:...
TRANSCRIPT
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Introduction to Metabolism
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Metabolism(The Acquisition and Utilization of Free Energy)
Catabolism: exergonic oxidationAnabolism: endergonic processes
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Endergonic Processes
• Mechanical Work
• Active Transport
• Biosynthesis
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Anabolism and Catabolism
exergonic
endergonic
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Roles of ATP and NADP+ in Metabolism
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ATP
Kinetic Stabilityof
Phosphoanhydride Bonds
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ATP
Adenosine
Ribose
Triphosphate
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Hydrolysis of ATP
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Phosphate Compounds
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Roles of ATP(Coupled Reactions)
∆Go’
(kJ/mol) ----------
Fructose-6-P + Pi ——> Fructose-1,6-bisP + H2O +13.3 ATP + H2O ——> ADP + Pi -30.5-------------------------------------------------------------------------------Fructose-6-P + ATP ——> Fructose-1,6-bisP + ADP -17.2
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Roles of ATP
• Early stages of nutrient breakdown
Glucose + ATP ——> Glucose-6-P + ADP
• Interconverson of nucleoside triphosphtes
NDP + ATP ——> NTP + ADP
Nucleoside Diphosphate Kinase
• Physiological processes
– Muscle contraction
– Active transport
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Roles of ATP
• Additional phosphoanhydride cleavages in highly endergonic reactons
(NMP)n + NTP ——> (NMP)n+1 + PPi
PPi + H2O ——> 2 Pi
Pyrophosphatase
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Sources of ATP
Phototrophs: photosynthesis
Chemotrophs: oxidation of organic compounds (e.g. carbohydrates, lipids, and proteins)
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Formation of ATP
• Adenylate Kinase reaction 2 ADP ——> AMP + ATP
• Substrate-level phosphorylation X–P + ADP ——> X–H + ATP
• Oxidative phosphorylation
• Photophosphorylation
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Substrate-Level Phosphorylation
Glyceraldehyde-3-PDehydrogenase
CHO
CHOH
CH2OPO3=
COOP
CHOH
CH2OP
1,3–Bisphosphoglycerate(1,3BPG)
+ NADH + H++ NAD+ + Pi
Glyceraldehyde-3-P(GA3P)
GA3P + NAD+ + H2O ——> 3PGA + NADH + H+ ² G°’ = –43.1 kJ/mol
3PGA + Pi ——> 1,3BPG + H2O ² G°’ = +49.3 kJ/mol
GA3P + NAD+ + Pi ——> 1,3BPG + NADH + H+ ² G°’ = +6.2 kJ/mol
COOP
CHOH
CH2OP
COOH
CHOH
CH2OP
3–Phosphoglycerate
+ ATP
1,3 Bisphosphoglycerate
+ ADP
PhosphoglycerateKinase
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Oxidative Phosphorylation
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Photophosphorylation
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Source of NAD(P)+, and other cofactors
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Source of NAD(P)+, and other cofactors
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NADP+
Nicotinamide Adenine Dinucleotide (Phosphate)
N
OCH2
OH OH
O
OCH2
OH OPO3=
AOP
O
O–
P
O
O–
O
CNH2
O
Nucleotide
Nucleotide
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Figure 14-1
Niacin
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Figure 14-11
Reduction of NAD+ or NADP+ to NADH or NADPH
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Metabolic Pathways
A ——> B ——> C ——> D ——> E
Metabolites
Enzymes
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Metabolic
Map
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Figure 14-3
Overview of
Catabolism
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Properties of Metabolic Pathways
• Separate Anabolic and Catabolic Pathways
• Steady-State
• Irreversible (overall): reversibility of individual steps
• First Committed (Exergonic) Step: others close to equilibrium
• Compartmentation (organelles & tissues): isoenzymes and transport
• Regulation (usually first committed step): often rate-limiting
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Potential Futile Cycles(Regulation)
CentralMetabolite Product/ Nutrient
Biosynthesis
Catabolism
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Steady State
Input OutputA A B B
Steady-State
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Thermodynamics of individual stepsA B
Go’ = -RTlnKeq
Not standard conditions or at equilibrium: G = Go’+RTln([B]/[A])
Three Physiological Conditions:Go’<<<<<<0 : G always negative
Example: ATP hydrolysisGo’>0 : near equilibrium, reversible,
direction depends on actual [B]/[A]Example: Most reactions
Go’>>>>>>0 : G always positive, must be coupledExample: Phosphorylation of Glucose
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Go’>0
Go’>0
Go’>0
Go’>0
Go’>0
Go’>0
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Go’>0
Go’>0
Go’>0
Go’>0
Go’>0
Go’>0
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Regulation of Metabolic Pathways
Specific Controls
General Controls
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Specific Controls
• Control of Enzyme Amount
– Constitutive Enzymes
– Inducible Enzymes
– Repressible Enzymes
• Control of Enzyme Activity
– Regulatory Enzymes
– Effectors (Ligands)
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General Controls(Integration of Cellular or Organism
Functions)
• Internal Effectors
– Catabolite Repression
– Energy Charge
– Reduction Potential
• External Effectors (e.g. hormones)
Significance: Efficiency and Flexibility!
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Types of Reactions
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Group Transfer Reactions
Y: + A—X Y—A + X:
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Phosphoryl Group Transfer
O
CH2OH
OH
OH
OHHO
O
CH2OPO3=
OH
OH
OHHO
- D- glucose–6–P(G6P)
- D- glucose(Glc)
ATP ADP
Mg2+
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Elimination Reactions
R C C R'
H
H
OH
H
C CR
H
H
R'+ H2O
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Isomerization Reactions(Intramolecular Hydrogen Shifts)
H C
CH O
O H
R
Aldose
C
CH O
O H
R
BH + C
CH O
O
R
H
H C
H
O
C O
R
H
Ketose
B: +
BH
B:
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Making C-C Bonds
C
H2C
COOH
COOHCH3 C S
O
CoAO
H2C
C
H2C
COOH
COOH
COOH
HO
Acetyl–SCoAOxaloacetate
Citrate
+CitrateSynthase
CoASH
Note: thioester
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Breaking C-C Bonds
CH2OPO32–
C
C
C
C
CH2OPO32–
O
HHO
OHH
OHH
CHO
CHOH
CH2OPO3=
CH2OH
C O
–D–fructose- 1,6- bisphosphate(FBP)
Glyceraldehyde–3–P[GA3P]
+
Dihydroxyacetone–P [DHAP]
Aldolase
CH2OPO3=
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Oxidation-Reduction Reactions
SH2 + NAD+ + H2O ——> S + NADH + H3O+
SH2: Reduced Substrate
S: Oxidized Product
NAD+: Electron Acceptor
FAD: Electron Acceptor
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Figure 14-11
Reduction of NAD+ to NADH
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Figure 14-12
Flavin Adenine Dinucleotide (FAD)
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Figure 14-13 part 1
Reduction of FAD to FADH2
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Figure 14-13 part 2
Reduction of FAD to FADH2
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One Electron Oxidation-Reduction Reactions
Fe3+ + Cu+ Fe2+ + Cu2+
ElectronAcceptor
ElectronDonor
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Half-Reactions
Fe3+ + e– Fe2+
Cu2+ + e–Cu+ (oxidation)
(reduction)
OxidationInvolves (e- of H:-)Loss
ReductionInvolves (e- of H:-)Gain
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Alcohol Dehydrogenase(Oxidation-Reduction Reaction)
H
C
C
H
O
H
H
H
H
N
R
C NH2
O
N
R
C NH2
OH H
C
CH3
OH
++
Ethanol
++
AlcoholDehydrogenase
H+
NADHNAD+
H+
H:–
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Experimental Approaches
to Metabolism
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Features of Metabolic Pathways
A ——> B ——> C ——> D ——> E
(1)Sequences and Energetics
(2) Enzymes and Mechanisms
(3) Control Mechanisms (Regulation)
(4) Compartmentation
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Elucidation of Metabolic Pathways
A ——> B ——> C ——> D ——> EMetabolic Inhibitors:
accumulation of intermediates
Biochemical Genetics: mutants
Pathway Labeling: isotopes
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Metabolic Inhibitors(Accumulation of Intermediates)
(e.g. Glycolysis)
Fluoride: (2-phosphoglycerate and consequently 3–phosphoglycerate)
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Biochemical Genetics(Mutants)
Natural Genetic DefectsManipulation of Microorganisms
Accumulation of Intermediates
Growth Requirements (auxotrophic mutants)
A ——> B ——> C ——> D ——> E
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Pathway Labeling
A* ——> B* ——> C*
Stable Isotopes
Radioisotopes
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Detection of Isotopes
• Stable Isotopes– Mass Spectrometry– NMR
• Radioisotopes– Proportional Counting (Geiger
Counter)– Liquid Scintillation Counting– Autoradiography
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Quantify Differential Expression
Condition 1 Condition 2
Sample Prep Sample Prep
Mix samples and detect
Quantify Differences
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Control of Expression
• Transcription:– Microarray
• Proteomics– 2D-SDS-PAGE– Isotope Coded Affinity Tag
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ICAT Chemistry
IAM
IAM
Biotin
Biotin
ReactiveGroup
(specific forcysteines)
AffinityTag
Isotopecode
(D or 13C)
LIGHT
HEAVY
ICAT = Isotope Coded Affinity Tag
Same behavior
chemically, but different
in mass.