Welcome to Biochemistry 440:
Introduction to Protein Structure & Metabolism
Prof. Justin Kollman (weeks 1-5)
Prof. Susan Brockerhoff (weeks 6-10)
Ms. Teri Alvarado (course coordinator)
course overview - mechanics, grading, etc. available on Canvas
Not registered? e-mail bioc440@uw.edu
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Biology follows the Laws of Thermodynamics
LIFE opposes ENTROPY
living systems are highly ordered and non-random
ORDER requires WORK and ENERGY input
2
biochemical reactions are arranged in complex,
interconnected pathways
biochemical reactions are arranged in complex,
interconnected pathways
+
glucose
free energy, G
Pi
glucose-6phosphate
many biochemical reactions are
NOT spontaneous
ATP
+H2O
Pi
ADP
-
free energy, G
linking biochemical activities to
drive reactions in the desired direction
ATP
ADP
-
Pi
free energy, G
+H2O
glucokinase
+
spontaneous
glucose
glucose-6phosphate
spontaneous = speedy
enzymes catalyze otherwise slow reactions
glucokinase
ATP
glucose
proteins are complex structures
fi
enzymes are highly speci c
proteins are dynamic machines
glucokinase
regulatory
protein
enzymes are exquisitely regulated
complex, interconnected pathways allow
cells and tissues to adapt to changing conditions
malfunctioning enzymes cause disease
understanding mechanisms can lead to
treatments
why do we care about biochemistry?
foundation of life
you can’t understand
cell biology, development, neuroscience, signalling, immunology,
microbiology, cancer biology, virology, systems biology…
without biochemistry
foundation of medicine
(almost) all drugs target proteins
most diseases result from aberrant proteins
protein
structure
enzyme
function
Prof. Kollman
weeks 1-5
metabolism
Prof. Brockerhoff
weeks 6-11
metabolism
energy metabolism
glycolysis & gluconeogenesis
glycogen metabolism
citric acid cycle
oxidative phosphorylation
photosynthesis
pentose phosphate pathway
Prof. Brockerhoff
weeks 6-11
Unifying themes of Biochemistry
Main concepts:
1.Biology follows the laws of thermodynamics
(review entropy, enthalpy, free energy)
2.Biochemical reactions are often linked
(review free energy)
3.Biochemical reactions are (almost) always catalyzed
(review activation energy vs. free energy)
4.Biochemical reactions occur in aqueous environments
(review properties of water)
21
pH: Why should we care?
Main concepts:
1.Biochemistry is pH-dependent
2.How buffers work
3.How blood is buffered
(CHEMISTRY review: weak acids/conjugate bases)
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pH: Protonation state is critical for biomolecules
low pH
high pH
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pH: Protonation state is critical for biomolecules
protein structure can be very sensitive to pH
R-bodies
light microscopy
cryo-electron microscopy
20 µm
0.5 µm
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Biological systems & pH
-
-
biochemical processes &
reactions are pH-dependent
pH in a given cell type,
organelle, or tissue must be
maintained near its optimum
for function
cytoplasm pH 7
lysosomes pH 4.8
gastric juices pH 1.5
bones - alkaline
25
Buffers in Biochemistry: in vivo & in vitro
concepts you should already KNOW (review!):
• strong vs. weak acids
• monoprotic vs. polyprotic acids
• weak acid/conjugate base
• pKa
• titration midpoint
[•
titration equivalence point (or stochiometric pt.)
not the same!
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How buffers work
biological buffers
weak acid/conjugate base pairs
resist pH changes from
acid (H+) or base (OH-) addition
used to maintain a specific pH
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How buffers work
biological buffers
weak acid/conjugate base pairs
resist pH changes from
acid (H+) or base (OH-) addition
used to maintain a specific pH
28
How buffers work: two equilibria
Observation: titration curve of an aqueous solution of weak
acid is steep at the ends and flat in the middle
flat region is: Buffering Region
titration of acetic acid
A-
weak acid
HA
HA
H + + A-
Henderson-Hasselbalch Eq’n
describes this behavior
tiny change
big change
Ka
conj.
base
pH = pKa + log([A-]/[HA])
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How buffers work
different buffers —> different pKa
pH = pKa + log([A-]/[HA])
[A-] = [HA]
pH = pKa
“midpoint”
Concept: further from pKa the larger the
fraction of molecules with - charge (high
pH) or + charge (low pH)
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example: the concept of equivalents
General form of net equilibrium:
HA + OH-
A- + H2O
1.0 equivalent = Total # mol (HA + A-)
Note: this is a CONSTANT for a given
buffer, so is knowable
Concept: the mole fraction of species Atells you how many equivalents of strong
base have been added to reach a given
pH, and vice versa
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How to make good buffer choices
Things you can know about a particular (biochemical) buffer
buffering range:
~ 2 pH units
+/- 1 of pKa
maximum buffering
capacity:
de ned by
([HA] + [A-])
fi
32
Biology’s buffers are dynamic
7.2-7.8
Physiological pH (blood) = 7.4
Large changes in blood pH are dangerous, so blood
pH must be maintained dynamically component [ ]s are changing
In vivo buffering systems: phosphate pKa = 6.8
carbonic acid —> pKa = 3.8
Ka
H2CO3 + H2O
H3O+ + HCO3-
weak
acid
conjugate
base
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Carbonic acid… not just another weak acid
Carbonic acid-Bicarbonate system: THREE equilibria
H3O+ + HCO3-
K1
H2CO3 + H2O
acid/base
K2
2 H2O + CO2
hydration/
deyhdration
K3
CO2
solution/
gas
Carbonic acid dissociates a proton to yield: bicarbonate
Carbonic acid reacts with water to yield:
CO2
Components are all products of metabolism, so
concentrations are: dynamic (changing)
H2CO3 in blood is in volatile equilibrium with LARGE reserve
of CO2 in lungs.
34
How do concentrations change in the body?
•
•
•
•
CO2 is produced by cellular respiration
CO2 is rapidly converted to bicarbonate
• catalyzed by carbonic anhydrase (an
enzyme)
erythrocyte
CO2 is exhaled in the lungs.
Rate of respiration controlled by brain
stem
generated
by resp’n
tissue
pH
lungs
pH
exhaled
35
The blood buffering system, simpli ed
Overall equilibrium (in solution):
H+ + HCO3-
K
CO2
Ka
where K = Ka • Kh
Kh
10-6.1 = 10-3.8 • 10-2.3
pK = 6.1
A modi ed HendersonHasselbach Eqn:
[HCO3-]
pH = pK + log
[CO2] (dissolved)
[HCO3-]
pH = 6.1 + log
[CO2] (dissolved)
fi
fi
36
The blood buffering system, simpli ed
Under normal conditions:
pH maintained
+/- 0.5 pH units
During exercise:
aerobic: resp’n produces more CO2
—> pH goes down
pH = 6.1 + log
[HCO3-]
[CO2]
anaerobic: lactic acid in blood
increases, more H+ —> pH goes down
Q: Why use a system with pKa = 6.1 instead of 7.4 in this system?
fi
37
pH: Why should we care?
Main concepts:
1.Biochemistry is pH-dependent
(know some concrete examples; concept of pKa)
2.How buffers work
(get comfortable with Henderson-Hasselbalch; be able to
work pH problems)
3.How blood is buffered
(understand linked equilibria; make predictions for
different situations)
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