BIOCHEMISTRY REVIEW Session I

1
BIOCHEMISTRY REVIEWSession I

• Bryan Mitton
• mittonb_at_ucmail.uc.edu

2
Biochemistry is almost over!
3
Todays Review

• 1) Amino Acids and Proteins
• 2) DNA and RNA
• 3) Glycolysis, Krebs Cycle, and ETC.
• Plus a 5 minute break between each section.

4
Amino Acids

• You need to know the basic structure of each AA,
  but not the pKas.
• A few AA facts
• Hydrophobicity is a function of the positional
  entropy of water. (Virtually always on test.)
• The only imino amino acid _______?
• Be able to calculate the isoelectric of any amino
  acid.
• Try Histidine pKa1 1.82
• pKa2 6.0
• pKa3 9.17

5
Isoelectric point

• The isoelectric point of an AA or protein is
  the pH at which there is NO NET CHARGE.

A B
C D
pKa1
pKa2
pKa3
6.0
1.82
9.17
6
Isoelectric point
6.0
9.17
1.82
Uncharged form. So average the pKa values
around it. (9.17 6)/2 7.6
7
Definitions

• Primary Structure
• Linear order of Amino Acids in a chain.
• Secondary Structure
• Comprised of beta pleated sheets, beta turns,
  alpha helices.
• Tertiary Structure
• How the secondary structures arrange themselves
  with respect to each other.
• Quaternary Structure
• Subunit-subunit interactions.
• What are the major physical forces that hold each
  structure together?

8
Forces

• Primary Structure Covalent
• Secondary Structure Hydrogen Bonding
• Tertiary Structure Hydrophobic Forces, Hydrogen
  Bonding, Salt Bridges, Van der Waals Forces, and
  Disulfide Bonds.
• The strongest covalent bonds are disulfide bonds.
• The strongest non-covalent bonds are salt
  bridges.
• The force that contributes the most to tertiary
  structure is HYDROPHOBIC forces.
• Hydrophobic residues put in core of protein and
  dictate stability.
• Quaternary Structure Same as tertiary.
• Q What AA is very likely to be found at beta
  turns?
• A Proline, as its imino structure allows for a
  tight turn.

9
Practice Qs.

• In the following peptide bond sketch, which atoms
  are coplanar?
• In an alpha helix, how many AA residues are there
  per turn? How long is one turn (the pitch)?
• What are prion diseases a result of?

10

• Which atoms in a peptide bond are coplanar?
• The C and N are both sp2 hybridized and so adopt
  a trigonal planar arrangement.

11

• In an alpha helix, how many AA residues are there
  per turn?

Answer 3.6 Amino acids, for a length of 5.4
Angstroms. The carbonyl of the 1st residue
hydrogen bonds with the amino group of the fourth.
12

• What causes prion diseases?
• Prion diseases result from accumulation of
  protein misfolding.
• The misfolded molecule is dubbed PrPSc.
• The misfolding of a PrPc molecule initiates a
  cascade of further misfolding
• PrPSc induces other properly folded to misfold.
  This polymerizes, causing cell damage disease.

13
Proteins

• 3 Proteins you need to know about
• Hemoglobin (myoglobin too)
• Collagen
• Elastin

14
Hemoglobin (Hb)

• Myoglobin 1 hemoglobin chain (almost).
• Myoglobin and each hemoglobin chain contains a
  heme group.
• Heme sits in an apolar pocket in the middle of
  each chain of hemoglobin/myoglobin.
• Heme is metabolized to bilirubin, the buildup of
  which causes of jaundice.
• Heme 1 iron atom plus a porphyrin ring.
• Porphyrin ring 4 pyrrole groups 4 methyl, 2
  vinyl, 2 propionates stuck onto it.
• Porphyrin ring coordinates with 4 Fe2 orbitals
  via nitrogen atoms.

15
Heme Group
Blue Nitrogen Black Carbon Red Iron
16
Oxygen binding

• The 5th coordination position of Fe2 is with a
  histidine.
• HIS 93 F8. This is the Proximal Histidine.
• Oxygen will be at the 6th spot.

17

• The Distal His is near where the oxygen binds.
  This is E7, or His 64.
• The distal is present to DECREASE Fe2 AFFINITY
  FOR CARBON MONOXIDE.
• Q What happens if Fe2 turns into Fe3?
• A It binds to water, becoming methemoglobin.

18
Nomenclature

• Adult hemoglobin is normally an a2b2 tetramer.
  This is called HbA.
• Also, 2 of total blood Hb is a2d2. This is
  HbA2.
• Fetally, here is the progression
• z2e2 a2g2 a2b2 plus a2d2
• A question about this was on my board exam
• Which one is fetal hemoglobin?
• A alpha 2 gamma 2.

19
Fetal vs. Adult Hb

• Important difference between gamma and beta
  chains
• BPG binds in a pocket that forms in the middle of
  all four chains when Hb is in the taut form.
• Recall TAUT low affinity for oxygen, so
  usually NO oxygen bound. RELAXED high affinity
  for oxygen, so usually oxygen is bound to Hb.
  More on this later.
• When bound, BPG lowers the affinity of Hb for
  oxygen because it stabilizes the taut form of Hb.
  
• Gamma chain a Serine is replaced with a
  Histidine, so BPG doesnt bind to fetal Hb very
  well.
• Thus, fetal Hb has HIGHER affinity for oxygen,
  because BPG doesnt bind to it and Hb remains in
  a relaxed conformation.

20
BPG Importance

• Q When happens to plasma BPG concentration at
  high altitude, and why?
• A Its concentration increases in the blood, so
  that hemoglobin spends more time in the taut
  conformation and lets go of oxygen more easily.
• Remember It lowers oxygen affinity by
  stabilizing the taut conformation of Hb.

21
Cooperativity

• Cooperativity Once the first oxygen is bound to
  Hb, it is easier for the other 3 to bind.

P50 27 torr
22
Cooperativity

• Compare the curves for myoglobin and hemoglobin.
• In the absence of BPG, Hb oxygen affinity curve
  looks like that for myoglobin.

23

• Hill coefficient 2.8 for Hb. Any Hill
  coefficient gt1 implies functional cooperativity
  among the molecules subunits.
• What is the Hill coefficient for myoglobin?

24
Hb Oxygenation

• When taut form gets oxygenated, Fe is pulled
  forward in heme group.
• This pulls on His 92 (the proximal Histidine, or
  F8) and this ends up breaking a hydrogen bond
  between Val 98 and Tyr 145.
• Breaking Val 98 Tyr 145 bond has two effects
• 1) An H-bond between His 146 Asp 94 is broken.
• 2) An H-bond between His 146 and a Lysine on the
  alpha chain is broken.
• As for the His 146 Asp 94 bond.

25
Bohr Effect

• Breaking the His 146 Lysine bond is how the
  beta chain tells the alpha chain it has picked up
  an oxygen.
• The His 146 Asp 94 bond is responsible for the
  Bohr effect.

26

• So, uh, what was the Bohr Effect again?
• Hemoglobins oxygen affinity is dependent upon
  the local pH and carbon dioxide level.
• It works like this
• If this bond is intact, Hb adopts Taut form.
• If this bond is broken, Hb adopts the Relaxed
  form.

27

• Thus, when oxygenated Hb enters a low pH zone,
  the His 146 gets protonated. It then forms the
  bond with Asp 94.
• When this bond is made, Hb will switch to the
  Taut form, and let go of oxygen.
• So protonation of His 146 is the key.

28

• Q List the 3 major variables/molecules that
  affect the affinity of Hb for oxygen.

29

• 1 factor pH
• 2 factor pCO2
• 3 factor BPG

30
Which graph is the one with LOWER oxygen affinity?
If we RAISE the following, WHICH WAY will the
curve shift? BPG pH pCO2
31
Hb

• Molecule can carry carbon dioxide, oxygen, and
  protons.
• 10 of all carbon dioxide in blood bound to 1st
  amino acid of Hb, Valine.
• Other 90 is carried as bicarbonate. The proton
  formed is part of Bohr effect.
• What enzyme catalyzes this reaction?

32
Sickle Cell Anemia

• The main problem in sickle cell anemia is a point
  mutation.
• Glutamate 6 is substituted with what residue?
• This was also on my board exam!
• When Hb is deoxygenated, this residue is exposed
  and causes polymerization of Hb.
• The chains form and deforms the cell, giving it
  the sickle shape.
• Having this mutation offers resistance to
  Plasmodium falciparum, the bug that causes
  malaria.
• Called HbS. Two bad chains SS, one bad one good
  AS (heterozygote).

33
Remember Glutamic Acid 6 is mutated to Valine,
and causes polymerization of Hb.
34
Thalassemia

• b-Thalassemia - Not enough b chains produced by
  cell, so a chains accumulate.
• a-Thalassemia Not enough a chains produced, and
  b chains accumulate.
• This disease is usually caused by a problem with
  splicing the mRNA isnt spliced correctly, so it
  gets destroyed.

35
Bone marrow expands in skull to make more RBC, so
you get this crew-cut appearance. Again a
thalassemia means you dont make a chains.
36

• Thats all for Hb!
• On to collagen.

37
Collagen

• The Amino Acid Sequence Gly-X-Y.
• Gly 33, Pro 20, 10 Ala. 5 lysine.
• Hydroxyproline and hydroxylysine are also part of
  primary structure.
• Prolylhydroxylase and lysylhydroxylase both
  require Ascorbic Acid, Vitamin C to work.
• These enzymes modify the individual polypeptides
  before they wrap up into the triple helix.
• Without Vitamin C, what disease do you get?

38
Scurvy, characterized by spontaneous bleeding
from joints and hair follicles.
39
Collagen The Vocab

• The confusing nomenclature of collagen
• One collagen polypeptide has a helical structure.
  This is the minor helix.
• Three polypeptides (three minor helices) wrap up
  to form the triple helix.
• A triple helix gets cleaved after it is exported
  from the cell at both the the N and C termini.
  After trimming, it is called tropocollagen.
• Tropocollagens line up to form fibrils.
• Fibrils line up to form the overall structure.

40

• Enzyme lysyl oxidase forms lysine cross-links in
  fibrils. Also requires vitamin C.
• Disulfide bonds form at both N and C termini
• At C termini, the disulfides form to help line up
  the 3 minor helices.
• At N termini, they form to stop intracellular
  fibrinogenesis.

41
Elastin

• Weird amino acids in it desmosine, etc.
• Has coiled-coil regions.
• No hydroxyproline or lysine.
• 1/3 ala val.
• Elastin is made of tropoelastin monomers.

42

• Take a 5 minute break!

43
II DNA and RNA
44
DNA and RNA

• Some facts
• Purines Adenine Guanine
• Pyrimidines Thymine Cytidine Uracil.
• Uracil found only in RNA.
• In double stranded DNA, G-C base pairing is
  stronger than T-A base pairing. Why?

45
(No Transcript)
46

• Chagraffs rule problems (T/F)
• In dsDNA
• If Tgt35, then Ggt15.
• Answer F.
• If Tgt35, then ATgt70, and Glt15.
• If A15 of the bases in one strand, G must 35
  of the bases in the whole ds molecule.
• Answer F. Who knows how many As there are in
  the other strand.
• If GC 40, then TG 50
• Answer T

47

• In ssRNA, if T24, then A24
• Answer F. It is single stranded, so there is no
  relationship.
• Lieberman Good Teacher
• Answer F
• Hes a GREAT teacher!!!

48
DNA and RNA synthesis

• RNA and DNA are made in the 5 to 3 direction.
• Be careful The template strand is read 3 to 5.
  Strands always run antiparallel.
• Drugs that stop HIV replication have some
  modification of the 3 OH group, such that
  phosphodiester bonds cannot be made after viral
  incorporation of these nucleotides. So, these
  drugs terminate viral replication.
• DNA Replication is semi-conservative.
• The parent strands are separated from each
  other, and after replication each parent strand
  is base-paired with newly-synthesized DNA.

49
Replication

• Replication begins at ori sites and proceeds
  bidirectionally.
• Helicase first unravels the DNA. Topoisomerase
  relieves tension developed during the unraveling
  at the other end.
• Leading and lagging strands form at each
  replication fork
• The following will demonstrate the names of the
  enzymes involved in prokaryotic DNA synthesis and
  the order in which they act.

50
Bacterial Replication
51

• Ligase connects the 5 end of primer/DNA to the
  3 end of completed DNA.
• Telomerase takes care of ends.
• Proofreading
• DNA pol I, II, and III all have 3 to 5
  exonuclease activity.
• DNA pol I is the only one with 5 to 3
  exonuclease activity.
• The parental DNA strand is identified by
  methylation, so that the wrong strand doesnt get
  changed.

52

• There are 3 DNA error correction systems
• Mismatch repair
• Base Excision Repair
• Nucleotide Excision Repair.

53
Mismatch Repair System

• In E. Coli, 3 Mut proteins recognize the
  mismatch.
• 1 of them makes a nick in the DNA 5 to the
  mistake on the unmethylated strand.
• This is called endonuclease activity.
• Exonucleases come in and remove a large piece of
  the DNA, and then Pol III fills in the space with
  the correct base pairs.

54
Base Excision Repair

• 2 Spontaneous Events. (Cell G0 phase)
• 1) Sometimes A or G just falls off the bond
  between the nucleotide and the riboses is
  spontaneously borken.
• 2) Sometimes the amine group falls off of
  cytosine, leaving a uracil behind.
• The uracil gets cut out when the cell detects
  this.
• Either way, a blank spot is left behind it is
  called the abasic site.
• AP Endonuclease comes in and nicks the DNA 5 to
  the abasic site. DNA pol I then comes in, excises
  the bases, and fills it in with good bases.

55
Nucleotide Excision Repair

• The system that fixes thymine dimers, which
  usually come of UV light exposure.
• Bacteria use UvrABC endonuclease to detect and
  nick the DNA near the thymine dimer.
• Pol I then excises the DNA section and
  simultaneously fills in gap.
• Xeroderma Pigmentosa humans with an inability
  to sufficiently repair DNA damage, especially
  thymine dimers, among other things. Prone to skin
  cancer, etc.
• There are at least 7 proteins associated with our
  DNA NER system Xp-1, 2, 3

56

• On to transcription and translation

57
Transcription

• Begins at the promoter, often called the TATA
  box, which is usually 10 bp away from first
  exon.
• First base transcribed is the 1 base, the one
  right beside it is -1.
• The sequence that the RNA polymerase reads is the
  Template strand.
• The template strand is the same thing as the
• NON-CODING strand.
• ANTI-SENSE strand.

58

1. The mRNA will have the same sequence as the DNA
   coding strand.
2. The mRNA will have the complementary sequence to
   the template strand.

59
Transcription

• Q There are 3 types of RNA polymerases
  Polymerase I, II, and III. What type of RNA do
  they each transcribe?
• A I rRNA II mRNA III tRNA
• 90 of all RNA is t or r.

60
Transcription

• In addition to the TATA box, two other DNA
  sequences affect how often pol jumps on the
  promoter.
• 1) Upstream regulatory elements lt200 BP away.
  (URE)
• 2) Enhancers/silencers anywhere in the entire
  genome.

61
Transcription

• Pol proceeds along DNA, making RNA 5 to 3.
• In bacteria, RNA pol II is a holoenzyme.
• It loses the s subunit when it binds DNA.
• Without s, pol cannot find promoter.
• The strand elongates, processively, like DNA pol
  III.
• Termination of transcription either
  rho-dependent or independent.
• If rho-independent, the mRNA being made forms a
  hairpin UUUUUUU-AAAAAAAA.
• If rho-dependent, the protein rho comes in and
  binds to the RNA to physically remove it from the
  DNA.

62
Transcription

• Bacteria have operons several genes in a row
  get transcribed to make a polycistronic message.
• Eukaryotes process out mRNAs individually.
• mRNA processing involves
• 5-5 7-methyl-Guanine cap on 5 end.
• Intron splicing.
• Poly A tail
• Something is modified at the Beginning, middle
  and end.

63
5-5 7-methyl-Guanine cap

• This cap is necessary for the mRNA to get out of
  the eukaryotic nucleus.
• Only mRNA gets capped.
• Has a weird 5 5 linkage 3 phosphates between
  the nucleosides.

64
(No Transcript)
65
Poly A tail

• The Poly A tail is made by Poly-A tail
  polymerase.
• The tail is not coded for in the genome.
• It is attached to the mRNA when transcription is
  over.
• Poly A tail is attached 10 to 30 bp after a
  AAUAAAAA sequence.

66
Splicing the removal of introns

• The 5 end of the intron to be removed is called
  the splice donor. 3 end is the splice acceptor.
• 1) 5 end of the intron is cleaved.
• 2) This is stuck onto an A residue about 20 bp in
  front of acceptor site. This makes a strange
  5-2 bond. This is called the lariat.
• 3) 3 end of intron cleaved, and exons are
  joined.
• All of this is done by snRNPs (small nuclear
  ribonucleoproteins).

67
The 25 bond forms here.
Lariat
68
Processing.

• mRNAs are capped and tailed BEFORE they are
  spliced.
• Introns can exist anywhere in the immature mRNA
  before or after start or stop codon.
• Note that in prokaryote, transcription and
  translation are simultaneous in eukaryote,
  processing occurs first in nucleus, then moves to
  cytosol.

69

• Find the intron

70
(No Transcript)
71
Transcription

• The genetic code is degenerate more than one
  codon codes for the same AA.
• 64 possible codons (43), but only 20 AAs.
• Think Many codons for one AA, but only one AA
  for a codon.

72
Translation Initiation

• Translation begins when a translation initiation
  factor (TIF) binds the first tRNA and a GTP.
• This whole complex binds the mRNA first.
• Next, the smaller ribosome subunit joins.
• Last, the larger ribosome subunit binds and the
  GTP is hydrolyzed.
• The first codon sits in the ribosomal P site, and
  the second sits in the A site.
• Overall Transcription initiation burns 1 GTP.

73
Translation Initiation

• First AA is usually Methionine, as its sequence
  is AUG, the start codon. This has a formyl
  group in bacteria, but not in eukaryotes.
• Hence, its called N-formyl Methionine.

74
mRNA Elongation

• Attaching an amino acid to a tRNA costs 1 GTP.
• Moving the ribosome down one codon also costs 1
  GTP (elongation factors use it).
• tRNA enters the ribosomal A site with its amino
  acid attached.
• At the P site, the previous AA gets covalently
  bonded to the first by the enzyme peptidyl
  transferase.
• What is the polarity of the growth of the peptide
  chain?

75

• The polypeptide chain grows from N terminal to
  the C terminal.
• Lame rhyming mnemonic for directionality
• 5 to 3, N to C

76
Wobble

• Wobble the tRNA anticodon (the 3 nucleotides of
  the tRNA that bind to the mRNA codon) can bind a
  few different sequences.
• The wobble base is the 3 end of codon, and the
  5 end of the tRNA.

77

• Which one is the wobble base position?

78
(No Transcript)
79
Gobbler
80

• tRNA can use Inosine as a base too.
• Dont memorize what wobbles with what just
  understand that last slide.

81
Stop!

• A stop codon exists, but there is no tRNA or
  amino acid for it.
• Ribosome simply disassembles.
• Termination costs 1 GTP, used by termination
  factors.
• To make a polypeptide n amino acids long, it
  costs
• 2n2 GTP.

82
Lac Operon
Under no lactose conditions, the I gene will be
transcribed and translated, and the I protein
binds the Operator site. With this protein bound,
polymerase cannot move beyond the operator. No X,
Y, or Z will be expressed.
83
Lac Operon

• When lactose is present, it binds to the I
  protein.
• The I protein now cannot bind the operator, and
  polymerase can transcribe the whole operon.
• The rate of transcription can be determined by
  cAMP levels. When cAMP binds to CRP
  (cAMP-Receptor Protein), or CAP (same thing),
  this complex strongly increases the affinity of
  pol II for the promotor.

84
Lac Operon

• cAMP levels increase as glucose decreases.
  Transcription rate of lacZ decreases in this
  order Lactose alone, Lac Glucose present,
  Glucose alone present.

85
Lac Operon

• Predict the results of these operon manipulations
  in the presence of lactose
• Overactive adenylyl cyclase.
• High cAMP, so lots of lacZ made.
• Mutated P site.
• Polymerase cannot bind, so no lacZ.
• Mutant lacZ.
• No lacZ made.

86

• Take a 5 minute break!

87
III Glycolysis, Krebs cycle, and the ETC
88
GLYCOLYSIS

• For all of metabolism
• Focus on regulation.
• Focus on rate-limiting steps.
• Ill give you the facts most likely to be tested.

89
1st Step Hexokinase

• Hexokinase Glucose Glucose-6P
• Works by induced-fit.
• Burns one ATP at a time.
• Irreversible enzyme.
• Negatively regulated by G6P.
• Phosphorylation traps Glucose in cell, so it
  cannot diffuse back out.
• Helped by GLUCOKINASE, which catalyzes the same
  reaction.
• Glucokinase has lower affinity for glucose, so
• 1) Has a high Km
• 2) Only works when Hexokinase is overburdened
• 3) Pushes glycolysis forward.

90
3rd Step. PFK-1

• PFK-1 Fructose-6P Fructose-1,6BP
• Major regulated step of glycolysis.
• Regulation of PFK-1 was on my board exam.
• Irreversible. Burns 1 ATP.
• It is activated by
• AMP and F26BP
• And inhibited by
• ATP and citrate.
• THINK ATP, citrate, vs. AMP and F26BP

91
Side reaction F26BP

• PFK-2 F6P Fructose-2,6BP.
• Fructose-2,6-bisphosphatase reverses this
  reaction.
• When PFK-2 is phosphorylated, PFK-2 is OFF, and
  F26BPase is ON.
• Opposite for dephosphorylation.
• Muscle isozyme not phosphorylatable, so this
  regulation only happens in the liver.

92
Side reaction F26BP

• If PFK-2 is phosphorylated, it is off, and
• F-2,6BPase will be on.
• Levels of F-2,6BP will drop.
• F-2,6BP normally activates PFK-1.
• So, in the liver, phosphorylation slows down
  glycolysis.

93
9th Reaction-Pyruvate Kinase

• Pyruvate Kinase Liver form.
• PEP and F16BP activate
• ATP and Alanine inhibit.
• Also phosphorylated to be
• OFF
• Since phosphorylation slows down PFK-1,
  phosphorylation of PK ought to also slow
  glycolysis.
• Muscle form not phosphorylatable. Just like
  PFK-2!!

94
Phosphorylatable Enzymes

• Enzymes all get phosphoryated after glucagon or
  epinephrine activates adenylyl cyclase, which
  then turns on PKA.
• PKA phosphorylates the relevant enzymes.
• When do things get phosphorylated?
• When you are hungry.
• When you are exercising.
• When you are afraid.

95

• Q Name the enzymes that catalyze the 3
  irreversible steps of glycolysis.
• Q Name the 3 major regulation points of
  glycolysis.
• Hint They are the same.

96

• Hexokinase
• PFK-1
• Pyruvate Kinase
• Phosphorylation is important it slows down
  glycolysis in the liver.
• What is the net yield of ATP from 1 molecule of
  glucose?
• Glucose 2NAD 2 pyruvate 2NADH 2ATP

97

• Carbon labeling
• Carbons 1 and 6 of glucose will become the top
  carbons of pyruvate.
• 2 and 5 are the middle carbons.
• 3 and 4 will become the ones on bottom.

98
Fructose and Galactose

• Fructokinase F F1P
• Next, F1P is cut into Dihydroxyacetone phosphate
  and glyceraldehyde.
• Triose kinase phosphorylates glyceraldehyde to
  G3P. These slide right into glycolysis.
• Fructose bypasses major regulated step of
  glycolysis!!!!!
• Phosphate wasting due to substrate level
  phosphorylations.
• Lactic acidosis also occurs because glycolysis is
  running extremely fast, which consumes NAD.
• Lactate is made from pyruvate to regenerate NAD.
  

99
Fructose and Galactose

• Galactose is made into Gal-1P by Galactokinase.
• Then an enzymes flips an OH group around to make
  it into Glucose 1P via UDP mechanism.
• Therefore, galactose does not bypass the major
  regulated step of glycolysis.

100
The Krebs Cycle

• Once Again, focus on regulation.
• Still responsible for names, products made by
  each reaction.

101
Inside the Mitochondrial Matrix

• First, pyruvate pumped into mitochondria via H
  symport or citrate antiport.
• Thats why/how citrate feeds back to stop
  glycolysis!
• Pyruvate goes to AcCoA irreversibly by Pyruvate
  DH, creating NADH.
• Cofactors of Pyruvate DH
• NAD
• FAD
• TPP (Vitamin B1, Thiamine Tri-Phosphate)
• 2 Lipoic Acids

102
Pyruvate DH

• Regulated so tightly because we cannot ever use
  the AcCoA carbons to directly make glucose again.
  
• Enzyme ON CoA, NAD, AMP
• Enzyme OFF AcCoA, NADH, ATP
• How do all these factors work?
• There is a kinase and a phosphatase on this huge
  complex when the above regulators are high/low,
  they simply influence the kinase and the
  phosphatase activity.
• Ultimately Phosphorylated OFF
• Dephosphorylated ON

103
Pyruvate DH

• Has to have
• NAD
• FAD
• TPP (Vitamin B1, Thiamine Tri-Phosphate)
• 2 Lipoic Acids
• Is turned on by
• CoA, NAD, AMP
• De-Phosphorylation
• Is turned off by
• AcCoA, NADH, ATP
• Phosphorylation

104
Krebs cycle

• No particular regulation
• There is usually little oxaloacetate present in
  the mitochondria, so this limits how fast the
  Krebs cycle proceeds forward.
• Isocitrate DH, alpha-ketoglutarate DH, and malate
  DH all make NADH.
• Succinate DH makes FADH2.

105
Electron Transport Chain

• High energy electrons move from complex to
  complex, driving Hydrogen into the intermembrane
  space.
• The hydrogen gradient drives ATP production.
  Called Proton-Motive Force

106
Intermembrane Space
H
H
H
H
I
IV
III
CoQ
e-
Cyt C
II
FAD
ADP
O2
NAD
Succinate
FADH
NADH
ATP
H2O
Fumarate
Matrix
107
Selected Inhibitors of ETC

• DNP is an uncoupling agent.
• It bonds to hydrogens in the intermembrane space,
  diffuses to the matrix, and lets them go.
• It ruins the H gradient, so electron transfer
  occurs in the absence of ATP production.
• Oligomycin blocks ATP synthase.
• Cyanide blocks complex IV.
• Rotenone blocks Complex I.
• Antimycin A blocks Complex III.

108

• Remember 2.5 ATP from NADH
• 1.5 ATP from FADH2
• This is because they enter the ETC at slightly
  different locations.

109
Summing it up

• Hexokinase G6P
• PFK 1 AMP and F26BP (ON)
• ATP and citrate. (OFF)
• PFK-2 Phosphorylation (OFF)
• Pyruvate Kinase PEP and F16BP (ON)
• ATP and Alanine
  (OFF)
• Pyruvate DH CoA, NAD, AMP (ON)
• AcCoA, NADH, ATP (OFF)
• Via Phosphorylation

110
GOOD LUCK!(See you in Pharmacology next year!)
Stay tuned for Chris Brubaker at 1 PM in this
room.