The Cell 4e

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Chapter 3
Cell Metabolism
Enzymes as catalysts Metabolic energy Synthesis
of cell constituents
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Why do we need catalysts?

• Biological reactions slow at body temperature and
  pressure
• - too slow for life w/o catalysts
• Enzymes increase reaction speed ( 106 times) to
  seconds ( milliseconds)
• - usually proteins
• - some RNAs (in ribosomes, telomere synthesis)
• - cells have thousands

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Whats so unique about enzymes?

• Increase reaction rate w/o being consumed/altered
• 2. Increase reaction rate w/o affecting chemical
  equilibrium
• E
• P R
• - equilibrium determined by thermodynamics
  of P and R

Energy diagram reveals role of enzymes - Bonds
in reactants (substrate) brought to higher
energy state transition state - Bonds can
then be broken and reformed in product
Catalysis interaction b/tw substrate and enzyme
in active site
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Enzymes react specifically w/ their substrates

• Interactions in active site based on
• H bonds
• Ionic bonds
• Hydrophobic interactions
• 2. Conformations of substrates altered
• - more toward transition state
• - amino acids in enzyme active site may bond w/
  reaction intermediates
• acidic/basic residues

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Serine proteases bind substrates viahydrophobic
ionic interactions
Preferential cleavage of peptide bonds adj. to
certain amino acids
3 aa in activ site Ser His Asp
Nature of pocket determines substrate specificity
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Model for catalysis chymotrypsin
Ser transfers H to His forming
tetrahedral transition state
Energetically favorable b/c His() now
interacts w/ Asp(-)
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Model for catalysis chymotrypsin
Peptide bond cleaved - His loses H to free
peptide - N terminus retained
Water donates H to His and OH- to substrate
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Model for catalysis chymotrypsin
2nd tetrahedral transition state
H transferred from His back to Ser
What can we learn from this? 1. Specificity E-S
2. Positioning of S in active site 3.
Requirement for active site residues
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Other participants in catalysis coenzymes

• enhance reaction rates
• carriers of chemical groups,
• some, prosthetic groups on proteins e.g. heme
  carries iron for binding O2
• some, small organic molecules
• unchanged by reaction, recycled

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Example of coenzyme function NAD/NADH
Nicotinamide adenine dinucleotide
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Enzymes regulated by feedback inhibition
2 types - positive - negative
Mechanisms - allosteric regulation -
competitive inhibition - phosphorylation
Activity affected by - temperature - pH
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Allosteric inhibition
Anything altering enzyme shape affects activity
Mechanisms - allosteric regulation -
competitive inhibition
Activity affected by - temperature - pH
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Phosphorylation of amino acids
Residues tyr, ser, thr - Kinases catalyze PO4
addition (100s / cell) - Phosphatases remove PO4
(100s/ cell)
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Energetics of biological reactions free energy
Chemical reactions obey laws of thermodynamics

• Descriptions of chemical reactions
• - Enthalpy
• Endothermic (?H) or exothermic (-?H)
• - Entropy
• Spontaneous (?S) or nonspontaneous (-?S)
• - Change in free energy encompasses both
• ?G ?H - T?S

Gibbs free energy named for J. Willard Gibbs
(1839-1903) -describes the change in energy of a
system (at constant T) If ?G lt0, spontaneous
?G gt0, nonspontaneous ?G 0, equilibrium
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Energetics of biological reactions free energy
Many biological reactions are nonspontaneous

• Have to couple them with spontaneous reactions
• - in which there will be an excess of free energy
• Overall, for both reactions, ?Glt0

A B ?G 10 kcal/mol C
D ?G -20 kcal/mol A C
B D ?G -10 kcal/mol
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ATP hydrolysis main cellular spontaneous
reaction

• Intracellular concentrations Pi 10-2 M, ATP gt
  ADP
• ATP ? ADP Pi ?G -12 kcal/mol

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The Generation of ATP from Glucose
Glycolysis ATP from glucose
The breakdown of carbohydrates, particularly
glucose, is a major source of cellular
energy Glycolysis - initial state in the
breakdown of glucose and is common to all
cells - oldest mechanism for energy
production In addition to producing ATP,
glycolysis converts two molecules of the coenzyme
NAD to NADH. Glycolysis takes place in the
cytosol
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3.11 Reactions of glycolysis
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• Complete oxidative breakdown of glucose yields ?G
  -686 kcal/mol
• Stepwise breakdown allows energy to be harvested
  for ATP synthesis
• Yield 4 ATP-2 ATP 2 ATP 2 NADH
• Regulated by feedback inhibition
• High ATP inhibits phosphofructokinase

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3.11 Reactions of glycolysis (Part 1)
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• - Hexokinase phosphorylates gluclose
• - Traps glucose inside cell
• - Inhibited by G-6-P

Hexokinase
Phosphohexose isomerase
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3

• Phosphofructose kinase
• Key regulator of glycolysis
• Inhibited by high ATP levels

Phosphofructose kinase
Aldolase
X
X Triosephosphate isomerase
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3
Phosphoglyceraldehyde dehydrogenase
3-Bisphosphoglycerate kinase

• NAD accepts e- from glyceraldehyde-3-phosphate
• Formation of 3-phosphoglycerate yields ?G
  -11.5 kcal/mol

Phosphoglycero- mutase
Enolase
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3

• Hydrolysis of phosphate from phosphoenolpyruvate
  yields ?G -14.6 kcal/mol
• Each glucose yields 4 ATP
• In absence of O2, NAD regenerated via
  fermentation

Pyruvate kinase
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Map of some biosynthetic pathways
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The Generation of ATP from Glucose
The Generation of ATP from Glucose
Pyruvate transport into the citric acid cycle

• Coenzyme A (CoA) - carrier of acyl groups in
  various metabolic reactions
• 1 carbon released as CO2
• Remaining 2 carbons transferred to CoA,
  then to citric acid cycle (TCA)
• TCA (or Krebs cycle) - central pathway in
  oxidative metabolism (mitochondria)

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TCA completes oxidation of glucose

• As the reactions cycle from citrate to
  oxaloacetate,
• high energy electrons are transferred to NAD
  and FAD

Electron accounting
Isocitrate ? ?-ketoglutarate, 2 e- to
NAD ?-ketoglutarate ? succinyl CoA, 2 e- to
NAD succinate ? fumarate, 2 e- to FAD
Carbon accounting

• 2 carbons enter as acetyl CoA
• 4 carbon oxaloacetate 6 carbon citrate
• 2 carbons released as CO2 (carbons from
  oxaloacetate are oxidized) 4 carbon succinyl
  CoA
• These 4 carbons regenerate starting material,
  oxaloacetate

http//www.sinauer.com/cooper/4e/animations0304.ht
ml
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3.13 The citric acid cycle, sung to the tune of
Rudolph the Red-nosed Reindeer
Citrate synthase
Malate dehydrogenase
Aconitase
Fumerase
Aconitase
Succinate dehydrogenase
Isocitrate dehydrogenase
Succinyl CoA synthase
?-ketoglutarate dehydrog.
ATP
ADP
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High Energy e- Donated to ETC
Fate of e- carried by NADH and FADH2
ETC
I
CoQ
III
2 e-
II
2 e-
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Transport of electrons from NADH
e- from NADH to O2 ?G -52.5 kcal/mol
-25.8 kcal/mol
-10.1 kcal/mol
-16.6 kcal/mol
4 H
4 H
2 H
NADH ox.
3 ATP
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Summary of ETC complexes

• Complex I 40 polypeptide chains, e- to flavin
  mononucleotide then the iron-sulfur center to
  CoQ
• Coenzyme Q, (ubiquinone), - small lipid-soluble
  molecule, carries electrons from complex I
  through the membrane to complex III, which
  consists of about ten polypeptides.
• Complex II - distinct protein complex, consists
  of four polypeptides, receives e- from the citric
  acid cycle intermediate, succinate (carried by
  FADH2)
• Complex III 10 polypeptides, e- goes from
  cytochrome b to cytochromec
• Cytochrome c - peripheral membrane protein on
  outer face of the mitochondrial inner membrane,
  carries electrons to complex IV
• Complex IV (cytochrome oxidase) 2 e- are added
  to oxygen, yields water

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11.10 Transport of electrons from FADH2
FADH2 ox.
2 ATP
4 H
2 H
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Figure 3.14 e- fall down the ETC
?G (free energy)
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Chemiosmotic Coupling
Chemiosmosis

• Chemiosmotic coupling - mechanism of coupling
  electron transport to ADP phosphorylation
• Brown fat (infants, humans in arctic climates,
  mammals) allows heat, instead of ATP to be
  generated via uncoupling proteins
• Certain disease states associated with
  uncoupling, e.g.
• Heart failure
• Diabetes
• e- transport through complexes I, III, and IV
  coupled to the transport of protons into the
  intermembrane space
• Proton gradient across inner membrane corresponds
  to about one pH unit (tenfold lower concentration
  of protons within mitochondria)

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11.11 The electrochemical nature of the proton
gradient
Phospholipid bilayer impermeable to ions
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Chemiosmotic Coupling

• 1961, Peter Mitchell proposed ATP synthesis via
  chemiosmosis
• Phosphorylation required intact membranes
• No high E intermediates between ETC and ATP found
• 1978 Nobel Prize
• Electrochemical gradient - difference in chemical
  concentration and electric potential across a
  membrane
• 0.14 volts across inner mito. membrane
• ATP synthase - transmembrane protein complex,
  couples the energetically favorable transport of
  protons across a membrane to the synthesis of ATP

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11.12 Structure of ATP synthase (complex V)

• F0 forms H channel
• E released as H diffuse down their
• concentration gradient
• E rotates F1, driving ADP phosphorylation

http//www.youtube.com/watch?v3y1dO4nNaKY
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Accounting for the oxidation of one molecule of
glucose

• 2 ATP
• 2 NADH
• 2 ATP
• 8 NADH
• 2 FADH2
• 34 ATP
• 38 ATP

Glycolysis
Pyruvate to Acetyl CoA The Citric Acid cycle
Electron transport chain
Total yield of ATP
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Energy from oxidation of fatty acids
1 16 C fatty acid 7 NADH 7 FADH2 8 Acetyl CoA 130
ATP
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Chemiosmotic Coupling
Energetics of biosynthesis

• Carbohydrates synthesized from CO2 (plants)
  pyruvate (animals)
• Calvin cycle-utilizes ATP and NADPH to synthesize
  glyceraldehyde 3-PO4 (then made into glc) from
  CO2
• -requires 18 ATP, 12 NADPH per glc
• Gluconeogenesis-reverse of glycolysis, some
  reactions reversible, others proceed only in
  direction of glc breakdown
• - energy requiring steps have different enzymes
• - requires 4 ATP, 2 GTP, 2 NADH

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Chemiosmotic Coupling
Carbohydrate synthesis in plants Calvin cycle
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Chemiosmotic Coupling
Gluconeogenesis
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Chemiosmotic Coupling
Polysaccharide synthesis
-ATP UTP provide energy -UDP-glc is activated
glc inter- mediate -UDP-glc donates glc to
poly- saccharide chain (E favorable)
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Chemiosmotic Coupling
Protein synthesis (not translation)
-N fixation requires ATP, few prokaryotes -prokary
otes, plants, fungi utilize NO3- -all organisms
incorporate NH3 into organic molecules -esp.
glutamate glutamine synthesis
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Chemiosmotic Coupling
Amino acid synthesis
-glc provides raw material -glu and gln donate
NH3 in synthesis of other AAs -requires energy
(ATP, NADPH plants prokaryotes ATP, NADH
animals) -glycolysis TCA intermediates provide
building blocks
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Chemiosmotic Coupling
Peptide bonds
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Chemiosmotic Coupling
Peptide bonds
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Chemiosmotic Coupling
Purine pyrimidine synthesis
-ribose-5-PO4 (from glc-6-PO4) -pur. pyr.
ribonucleotides -ribonucleotides ?deoxyribo-
nucleotides