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Transcript
Welcome to Class 8
Introductory Biochemistry
Announcements / Reminders
Midterm TA led Review Sessions
Sunday, February 28 from 6-8 pm
Location: Smith-Buonanno 106
Office Hours
Prof Salomon: by appointment
TAs: See canvas for details
Midterm 1 is Tuesday March 1 at 1 pm
Location: Last names beginning with A-M Macmillan 117
Last names beginning N-Z Barus and Holley 168
1
Class 8: Outline and Objectives
Redox reactions
l  Oxidation states of carbon
l  Relationship between ∆G and ∆E
l  Electron carriers NADH, NADPH, FAD, FMN
l  Where does the energy come from?
l  Overview metabolism, catabolism, anabolism
l  Glycolysis
l  Pathway
l  Regulation
l  Substrate channeling
l  Fermentation
l  Lactate
l  Ethanol
l  Pentose phosphate pathway
l  Fructose and galactose metabolism
l 
Many enzyme reactions involve a change
in the oxidation state of the substrate
2
Redox Reactions
Electrons are transferred in reduction-oxidation reactions.
Redox reactions require an electron donor (reducing agent)
and an electron acceptor (oxidizing agent).
Therefore, two simultaneous reactions occur in a redox process.
e- affinity
stronger
oxidants
stronger
reductants
The reduction potentials of redox half-reactions under standard conditions
can be tabulated, like ΔG' º values for other reactions.
The strongest oxidant is O2 and the strongest reductant is a small Feprotein called ferredoxin.
3
stronger
oxidants
stronger
reductants
The E' º value is for a half reaction under standard conditions (all components
at 1 M except H+ and H2O). The units of E' º is volts (V).
Standard reduction potentials, E' º, are relative to that of hydrogen (E' º ≡ 0).
Measurement of the standard reduction potential of a redox pair
Figure 13-23
4
Standard Reduction Potentials E’°
The reduction potentials of redox couples under standard conditions can be
tabulated, like ΔG' º values for other reactions.
The E' º value is for a half reaction under standard conditions (all components
at 1 M except H+ and H2O). The units of E' º is volts (V).
The tabulated reduction potentials are for the reduction reaction:
Oxidized form + electrons → Reduced form
E' º (Volts)
Standard reduction potentials, E' º, are relative to that of hydrogen (E' º ≡ 0).
For an actual redox reaction, a reduction must be accompanied by a oxidation
so that electrons are not “left over” or deficient.
For any complete redox reaction, ΔE' º = E' º for the substance being reduced
(the oxidant) minus E' º for the substance being oxidized (the reductant).
stronger
oxidants
stronger
reductants
The tabulated reduction potentials are for the reduction reaction: Oxidized form +
electrons → Reduced form
Example:
2Fe3+ + Ethanol →2Fe2+ + Acetaldehyde + 2H+
ΔE' º = 0.771 – (–0.197)= 0.968 V
Note that the number of atoms or molecules involved, and the number of electrons
transferred, don’t enter into these calculations.
5
Oxidation states of carbon in the biosphere
C bonding
electrons
C bonding
electrons
Figure 13-22
increasing electronegativity: H < C < S < N < O
the more electronegative atom “owns” the bonding electrons
Concentration dependence of E
Like ∆G, the reduction potential E of a half cell is concentration dependent:
E = E' º +
[electron acceptor A+]
RT
ln
nF
[electron donor A]
ΔE = (E2 – E1) V
E = E1 V
2H+/H2
+
–
E = E2 V
2A+/2A
If [A+] = [A], E = E' º.
If [A+] > [A], E > E' º.
If [A+] < [A], E < E' º.
Example: The ratio NAD+/NADH is kept high in the cell. This makes E more positive
than E' º (makes the couple a stronger oxidant than if [NAD+] = [NADH]) and favors
the reaction direction NAD+ + H+ → NADH (ΔG is more negative than ΔG' º)
6
Standard Reduction Potential Differences
are Mathematically Related to the
Standard Free Energy for the Reaction
2Fe3+ + Ethanol → 2Fe2+ + Acetaldehyde + 2H+
ΔE' º = 0.771 – (–0.197) = 0.968 V
ΔG = –nFΔE
n = the number of electrons transferred in the reaction
F = The Faraday Constant: 96.5 kJ/V-mol
For the above reaction, ΔG' º = –(2)(96.5 kJ/V-mol)(0.968 V) = –186.8 kJ/mol.
Note that the number of electrons exchanged does enter into the calculation of
ΔG' º from ΔE' º.
Note that a positive ΔE' º corresponds to a negative ΔG' º (exergonic).
stronger
oxidants
stronger
reductants
Electrons are transferred by cofactors acting as electron carriers
• Very important biological redox cofactors are the pyridine nucleotides and flavin
nucleotides.
• The reduced forms of these cofactors are relatively strong reductants.
7
Pyridine Nucleotide Coenzymes
Niacin (vitamin B3)
Nicotinamide ring
Nicotinamide Adenine Dinucleotide (NAD)
Nicotinamide Adenine Dinucleotide
Phosphate (NADP)
NAD(P)+ = oxidized form
NAD(P)H = reduced form
Figure 13-24
Riboflavin (vitamin B2)
Flavin nucleotide cofactors
Flavin Mononucleotide (FMN) and
Flavin Adenine Dinucleotide (FAD)
FMN, FAD = oxidized forms
FMNH2, FADH2 = reduced forms
Figure 13-27
8
Coenzymes NAD and NADP
The reduced nicotinamide ring has a characteristic near-UV
absorption near 340 nm, which can be used to follow the
progress of the enzymatic reaction in kinetic experiments.
Figure 13-24b
Four ways to transfer electrons
l 
l 
l 
l 
Directly as electrons (Fe2+ + Cu2+ ↔ Fe3+ + Cu+)
As hydrogen atoms (AH2 + B ↔ A + BH2)
—
As a hydride ion :H (CH3CH3 + NAD+ ↔ CH2=CH2 + NADH + H+)
Through direct combination with oxygen (RCH3 + 1/2 O2 ↔ RCH2OH)
For most biological molecules, the unit of oxidation and reduction is two
reducing equivalents, i.e., two electrons, i.e., pairs of electrons are gained
or lost in each redox reaction.
In biological systems, oxidation is often synonymous with dehydrogenation
(loss of hydrogen, note that there is no oxygen involved),
i.e. gain of a double bond between carbon atoms or change of an alcohol to
a carbonyl oxygen in an organic molecule.
2 Fe3+ +
2 Fe2+ +
+ 2 H+
9
Several ways of expressing redox reactions
NAD+ + H+ + 2e–
NAD+ + 2[H]
NADP+ + 2[H]
NAD(P)+ + 2[H]
FMN + 2H+ + 2e–
FAD + 2[H]
NADH
NADH + H+
NADPH + H+
NAD(P)H + H+
FMNH2
FADH2
Overview: Catabolism and anabolism
metabolism: meta = change; bolism ≈ throwing
ana = up, back, again
reduction
cata = down
oxidation
Part II, Figure 3
10
Catabolic pathways converge, anabolic pathways diverge
Part II, Figure 4
Three stages of cellular respiration
Glycolysis “splits” sugars and partially
oxidizes the products, generating substrates
for complete oxidation to CO2, and also
generates some ATP and reduced cofactors.
(In fermentations, the reduced cofactors are
re-oxidized in non-energy-generating
reactions.)
Glycolysis: glyco = sugar; lysis = splitting
The citric acid cycle completely oxidizes the
products of glycolysis to CO2 and generates
reduced cofactors as well as some ATP.
Respiratory re-oxidation of the reduced
cofactors generated in the above stages is
coupled to the synthesis of large amounts of
ATP.
Figure 16-1
11
Class 8: Outline and Objectives
l  Redox reactions
l Oxidation states of carbon
l Relationship between ∆G and ∆E
l  Electron carriers NADH, NADPH, FAD, FMN
l Where does the energy come from?
l  Overview metabolism, catabolism, anabolism
l  Glycolysis
l Pathway
l Regulation
l Substrate channeling
l  Fermentation
l Lactate
l Ethanol
l  Pentose phosphate pathway
l  Fructose and galactose metabolism
Oxidation states of carbon in the biosphere
C bonding
electrons
5 4
4
4
4
C bonding
electrons
3
Sugars have a composition
like formaldehyde, (CH2O)n
CH2O + H2O → CO2 + 4H+ + 4e–
Figure 13-22
12
Fire: NOT stepwise oxidation of glucose
Glycolysis: stepwise oxidation of glucose
● 
Oxygen has a higher affinity for electrons than glucose and the various
electron carriers (e.g. NADH).
● 
Therefore, the transfer of electrons from these molecules to O2 is
energetically favorable.
● 
Glucose + 6 O2
● 
Only 30-50 kJ/mol are required to form one molecule of ATP from ADP + Pi
● 
Stepwise oxidation of glucose converts electron flow into usable energy.
The energy released in some steps can be captured by coupling the step to
ATP synthesis, or by temporarily storing the electrons in a molecule (e.g.,
NADH) whose re-oxidation can be coupled to ATP synthesis.
6 CO2 + 6 H2O
∆G´o = –2840 kJ/mol
= –473.3 kJ/mol C
13
Glycolysis: Part I
Figure 14-2-1
Glycolysis: Part II
Figure 14-2-2
14
1. Formation of glucose 6-phosphate
1
Glucose + Pi ↔ Glucose 6-P
ATP ↔ ADP + Pi
Glucose + ATP ↔ Glucose 6-P + ADP
ΔG'0 = 13.8 kJ/mol
ΔG'0 = –30.5 kJ/mol
ΔG'0 = –16.7 kJ/mol
The large negative ΔG’° for ATP hydrolysis drives the reaction.
Coupled reactions drive endergonic processes.
1. Formation of glucose 6-phosphate
Importance of phosphorylated intermediates:
●  Negative charge traps intermediates inside the cell, even if the concentration
outside is much lower.
●  Phosphoryl groups conserve energy.
●  The binding energy resulting from enzyme interactions with phosphate
groups helps to lower the activation energy and increases the specificity of
reactions.
15
2. Isomerization and second phosphorylation
2
Ketose
Aldose
3
Enzyme-limited vs. substrate-limited reactions
1
2
3
Enzyme-limited
reaction (far from
equilibrium)
Substrate-limited
reactions (at or
near equilibrium)
Enzyme-limited
reaction (far from
equilibrium)
For some steps in glycolysis, the substrate/product
ratios are near the equilibrium ratios, because the
involved enzymes are relatively fast or abundant.
→ flux is substrate-limited.
Example: steps 2, 4
For a step that is catalyzed by a relatively slow or
scarce enzyme, the substrate/product ratio is
greater than the equilibrium ratio.
→ flux is enzyme-limited.
Example: steps 1, 3
4
Lehninger 3rd Ed., Figure 15-16
16
Regulation of metabolic pathways
Metabolic flux must be controlled:
● 
the demand for ATP production in muscle may increase 100-fold in a
few seconds in response to exercise
● 
relative proportions of carbohydrate, fat and protein in the diet vary
from meal to meal
● 
the supply of fuels obtained in the diet is intermittent (between meals,
starvation)
Regulation of metabolic pathways
Most enzymes in a pathway operate near their equilibrium, but some
enzymes that are stategically located operate far from equilibrium.
Characteristics of enzymes that are regulated:
● 
They control the rate of the respective pathway and whether it is
turned on or shut off.
● 
They catalyze reactions that are out of equilibrium and which are
enzyme-limited (valve function).
● 
They catalyze highly exergonic (effectively irreversible) reactions, thus
driving the pathway forward.
● 
They differ in a catabolic vs. anabolic pathway.
17
The two ATP-requiring steps of glycolysis are regulated
Figure 14-2-1
Allosteric control of enzyme activity: Hexokinase
Allosteric
site
Glucose-bound form: induced fit
Hexokinase is allosterically inhibited by its own product, glucose 6-phosphate.
Figure 6-25
18
PFK-1 regulation is complex
ATP is a required substrate, but at
high concentration, ATP can also
bind to an allosteric site and inhibit
PFK-1. This inhibition is relieved by
AMP, which competes with ATP for
binding at the allosteric site.
Figure 15-16b,c
4. Aldol cleavage (the literal glycolytic reaction)
Even though ∆G' º > 0, the reaction proceeds in the forward direction
because the reaction products are removed quickly by later steps, “pulling”
the reaction in the direction of cleavage. ∆G < 0.
19
5. Isomerization
Ketose
Aldose
Even though ∆G' º > 0, the reaction proceeds in the forward direction
because the reaction products are removed quickly by later steps, “pulling”
the reaction in the direction of cleavage. ∆G < 0.
Triosephosphate isomerase: a “perfect enzyme”
•  Limitation 1: a catalyst cannot affect the position of the reaction equilibrium
•  TIM accelerates the isomerization by a factor of 1010 compared to the
chemical reaction
•  Limitation 2: a catalyst cannot catalyze an interconversion faster
than the substrate can ‘find’ the enzyme in solution
•  the kcat/KM ratio for TIM is 2 x 108 M-1 s-1, which is close to the
diffusion limit of a small molecule in solution
20
The fate of the hexose carbon atoms
Figure 14-7
Glycolysis: Summary of Part I
In this phase, a glucose molecule is converted to two triose phosphate
molecules, at the expense of (driven by) the hydrolysis of two ATP molecules.
Figure 14-2-1
21
Glycolysis: Part II
Figure 14-2-2
The first substrate-level phosphorylation uses two enzymes
6
7
22
Oxidative Phosphorylation of Glyceraldehyde 3-P
6
3-P-glycerate + 2 H+ + 2 e– ↔ Glyceraldehyde 3-P
E'o = -0.55 V
+
+
–
o
NAD + H + 2 e ↔ NADH
E' = –0.32 V ΔG' 0 = -2(96.5)(-0.32+0.55)
ΔG' 0 = - 44.4kJ/mol
3-P-glycerate + Pi ↔ 1,3-bis-P-glycerate
ΔG' 0 = 50.7 kJ/mol
Glyceraldehyde 3-P + Pi + NAD+ ↔ 1,3-bis-P-glycerate + NADH + H+ ΔG' 0 =
6.3 kJ/mol
ΔG'
o
= –nFΔE' o
The very large negative ΔG' 0 for glyceraldehyde 3-P oxidation drives the reaction
7
The first substrate-level ADP phosphorylation
1,3-bis-P-Glycerate ↔ 3-P-Glycerate + Pi
ADP + Pi ↔ ATP
1,3-bis-P-Glycerate + ADP ↔ 3-P-glycerate + ATP
ΔG' 0 = –49.0 kJ/mol
ΔG' 0 = 30.5 kJ/mol
ΔG' 0 = –18.5 kJ/mol
The large negative ΔG' 0 for 1,3-bis-P-glycerate hydrolysis drives the reaction
23
1,3-BPG is a high-energy compound
Figure 13-19
Why is hydrolysis of 1,3-BPG so exergonic?
1. 
The reaction product 3-P-glyceric acid is a strong acid, which immediately
ionizes at physiological pH.
2. 
Resonance of the ionized reaction product 3-P-glycerate distributes the
negative charge over 2 oxygen atoms, which stabilizes the ionized form
and effectively lowers the concentration of the immediate hydrolysis
product.
Figure 13-14
24
The coupled reaction [6 + 7] is overall exergonic
Glyceraldehyde 3-P + ADP + Pi + NAD+ → 3-P-Glycerate + ATP + NADH + H+
Overall ∆G' º = 6.3 + (–18.5) = –12.2 kJ/mol
Under cellular conditions, steps 6 and 7 are reversible.
6
7
Substrate channeling between reactions 6 and 7
Protects 1,3-bisphosphoglycerate
from spontaneous hydrolysis
Ensures that reactions 6 and 7
remain closely coupled
Lehninger 3rd Ed., Figure 15-8
25
2,3-BPG is an intermediate in step 8
8
9
The second substrate-level phosphorylation
10
26
Like 1,3-BPG, PEP is a high-energy compound
Figure 13-19
PEP hydrolysis
PEP hydrolysis is driven by tautomerization of the immediate hydrolysis
product (enol form) to the more stable keto form, which lowers the effective
concentration of the immediate hydrolysis product.
About half of the released energy (∆G' º = –61.9 kJ/mol) is captured in the
formation of ATP (∆G' º = –30.5 kJ/mol), the rest (net ∆G' º = –31.4 kJ/mol)
constitutes a driving force to “pull” the reaction forward.
Figure 13-13
27
Glycolysis: Summary of Part II
In this phase, the initial
two ATP molecules that
were hydrolyzed in Part
I are regenerated, and
two more ATP plus two
NADH molecules are
formed for each
molecule of glucose.
Figure 14-2-2
Steady-state concentrations of glycolytic intermediates in erythrocytes
Metabolite
Glucose
Glucose 6-P
Fructose 6-P
Fructose 1,6-bis-P
Dihydroxyacetone-P
Glyceraldehyde 3-P
1,3-bis-P-glycerate
2,3-bis-P-glycerate
3-P-glycerate
2-P-glycerate
P-enol-pyruvate
Pyruvate
Lactate
ATP
ADP
Pi
Concentration (mM)
5.0
0.083
0.014
0.031
0.14
0.019
0.001
4.0
0.12
0.030
0.023
0.0051
2.9
1.85
0.14
1.0
From Minakami, S. and Yoshikawa, H. 1965. Biochem. Biophys. Res. Comm. 18: 545.
28
The standard free energies of
some of the steps of glycolysis
are positive, even though the
overall standard free energy is
negative.
At the actual cellular concentrations
of the intermediates, the free
energies of all of the steps are
either negative or close to zero.
Garrett and Grisham, Figure 18-31
Calculation of the overall energetics of glycolysis
First, calculate the energy released on complete oxidation of pyruvate:
Glucose (C6H12O6) + 6 O2 ↔ 6 CO2 + 6 H2O
2 Pyruvate (C3H4O3) + 2 NADH + 2 H+ ↔ Glucose + 2 NAD+
2 NAD+ + 2 H+ + 4 e– ↔ 2 NADH
E'o = –0.320 V
2 H2O ↔ O2 + 4 H+ + 4 e–
E'o = –0.816 V
2 Pyruvate + 5 O2 ↔ 6 CO2 + 4 H2O
ΔG' 0 = –2840 kJ/mol
ΔG' 0 = 146 kJ/mol
ΔG' 0 = 123.5 kJ/mol
ΔG' 0 = 315.0 kJ/mol
ΔG' 0 = –2255.5 kJ/mol
Then use the value for the complete oxidation of 2 pyruvates to calculate the overall energetics of
glycolysis:
Glucose + 6 O2 ↔ 6 CO2 + 6 H2O
ΔG' 0 = –2840 kJ/mol
6 CO2 + 4 H2O ↔ 2 Pyruvate + 5 O2
ΔG' 0 = 2255.5 kJ/mol
2 ADP + 2 Pi ↔ 2 ATP + 2 H2O
ΔG' 0 = 61 kJ/mol
2 NAD+ + 2 H+ + 4 e– ↔ 2 NADH
E'o = –0.320 V
ΔG' 0 = 123.5 kJ/mol
2 H2O ↔ O2 + 4 H+ + 4 e–
E'o = –0.816 V
ΔG' 0 = 315 kJ/mol
Glucose + 2 ADP + 2 Pi + 2 NAD+ ↔ 2 Pyruvate + 2 ATP + 2 H2O + 2 NADH + 2 H+ ΔG' 0 = –82 kJ/mol
(Values from Lehninger)
Of the energy released in the partial oxidation of glucose to pyruvate, 61/2840 = 2.1% is
captured as ATP. If O2 is available and can be used, another 438.5/2840 = 15.4% is
potentially obtainable from re-oxidation of NADH, and an additional 2255.5/2840 = 79.4%
is potentially obtainable from the complete oxidation of pyruvate.
29
Reoxidation of NADH is critical
Glucose + 2 ADP + 2 Pi + 2 NAD+ →
2 Pyruvate + 2 ATP + 2 H2O + 2 NADH + 2 H+
ΔG'º = –82 kJ/mol
Compared to the amount of glucose that is converted
to pyruvate, there are tiny amounts of NAD+ in cells.
Glycolysis would quickly grind to a halt unless NADH
is re-oxidized to NAD+.
Class 8: Outline and Objectives
l  Redox reactions
l Oxidation states of carbon
l Relationship between ∆G and ∆E
l  Electron carriers NADH, NADPH, FAD, FMN
l Where does the energy come from?
l  Overview metabolism, catabolism, anabolism
l  Glycolysis
l Pathway
l Regulation
l Substrate channeling
l  Fermentation
l Lactate
l Ethanol
l  Pentose phosphate pathway
l  Fructose and galactose metabolism
30
Fates of Pyruvate: Aerobic and anaerobic pathways
muscle,
microorganisms
yeast
Lactic acid and alcohol fermentation
Both processes regenerate NAD+ to allow continued glycolysis.
muscle,
microorganisms
yeast
31
Fermentation and the Pasteur Effect
In the absence of oxygen, the ATP generated during glycolysis is the sole energy
derived from partial glucose oxidation to pyruvate.
Problem that needs to be solved:
NAD+ has to be regenerated to allow continued glycolysis.
Solution:
Reduce pyruvate, e.g. to lactate or ethanol,
using the NADH that was generated during
glycolysis.
This does not require or generate energy (ATP).
Fermentation does not capture very much of the energy that is potentially available
from the complete oxidation of glucose.
Energy available from complete glucose oxidation: –2840 kJ/mol
Energy required to form two mols of ATP: 2 x 30.5 = 61 kJ/mol of glucose (2.1 %).
In organisms that can ferment in the absence of O2 and respire in the presence of
O2, the rate of anaerobic glucose consumption is much higher (Pasteur effect in
yeast).
Major pathways of glucose utilization
and NADPH
Figure 14-1
32
Pentose phosphate pathway - Overview
Figure 14-21
Pentose phosphate pathway
Figure 14-22
33
Recycling of pentose phosphates in
nonoxidative reactions
(6)
6
2
2
6→5
(2)
(4)
4
(2)
2
(3)
(2)
2
3
(2)
(2)
(1)
(2)
2
2
Figure 14-23a
Recycling of pentose phosphates in
nonoxidative reactions
Figure 14-23b
34
Feeder pathways (convergence)
Figure 14-11
Fructose catabolism
liver
muscle, kidney
glycerol
triacylglycerides
glycerol-3-phosphate
phospholipids
35
①
Galactose catabolism
1. Galactokinase phosphorylates galactose
to form galactose-1-P
④
Phosphoglucomutase
②
③
NADH
NAD+
Glucose 6-phosphate
2. Galactose-1-P is exchanged for glucose-1P of UDP-glucose (a sugar-nucleotide).
UDP (uridine diphosphate) functions as a
coenzyme-like carrier of hexose groups
3. UDP-galactose is isomerized to UDPglucose by an epimerase that contains a
bound NAD+. Mechanism involves
stereoselective reduction of a C4 oxidized
intermediate in a 2 step process.
4. Glucose-1-P is isomerized to glucose-6-P
Figure 14-13
36