Symposium on Protein Misfolding Diseases
Biochemistry & Molecular Biology website
May 1-2, 2007
University of Massachusetts Amherst
Tuesday, May 1
1:00-1:30pm Registration
1:30-3:30pm Panel discussion: Therapeutic approaches to protein misfolding diseases
Moderator: Jonathan King, MIT
Panel members: Mark Findeis, Satori Pharmaceuticals; Richard Labaudinaire, FoldRx;
Pedro Huertas, Harvard-MIT Division of Health Sciences and Technology; Tim
Edmunds, Genzyme
4 pm Special Seminar by Jeffery Kelly, Scripps; "Understanding and ameliorating age onset
neurodegenerative diseases“ (A student-invited seminar hosted by the Chemistry-Biology
Interface Program)
Wednesday, May 2
8:30-9:00am Registration
9-9:50am Valina Dawson, Johns Hopkins; "Proteins behaving badly: Clues to Parkinson's
disease"
9:50-10:40am Ron Wetzel, Univ. of Pittsburgh; "The long and short of neurotoxic
polyglutamine sequences"
11:00-11:50am Rick Morimoto, Northwestern; "Stress and misfolded proteins: Insights into
mechanisms of aging and neurodegenerative disease"
11:50-2:00pm Lunch/posters
2:00-2:50pm Ron Kopito, Stanford; "The ubiquitin system in protein homeostasis"
2:50-3:40pm Byron Caughey, Rocky Mtn. Labs.; "Prion protein aggregation and disease"
4:00-5:00pm Susan Lindquist, the 2007 John Nordin Lecturer, Whitehead Inst.; "Yeast as a
discovery platform for protein folding diseases"
5:00-6:00pm Reception
The symposium will take place in the Murray D. Lincoln Campus Center at the University of
Massachusetts Amherst in the Pioneer Valley of Western Massachusetts.
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© 2004 New Science Press Ltd new-science-press.com
In association with
BioMed Central
1
Catalysis and
control of protein function
Structure and mechanism in protein science. A. Fersht.
Chapter 3A and 3B.
Petzco & Ringe Chapter 2.6 to 2.9.
The concept of steady state
In dynamic systems
Voet Biochemistry 3e Page 478
© 2004 John Wiley & Sons, Inc.
[S0] >> [ET]
pre-steady state
2
Michaelis-Menten Mechanism
k1
E+S
ES
k2
P + E
k-1
v0 = k2 [ES] = k2 [E]T [S] / (KM + [S]) = Vmax [S] / (KM + [S])
KM is analogous to the dissociation constant of the
Michaelis complex
k << k (rapid equilibrium)
2
-1
KM = k-1/k1 = KdS
At low [S]
At high [S]
v ∝ [S]
v = Vmax = ET kcat
Michaelis-Menten Equation
The equation holds for many mechanisms,
not only for the M-M mechanism.
(e.g. Briggs-Haldane kinetics: KM > KdS)
v0 = Vmax [S] / (KM + [S])
Vmax = kcat [ET]
3
The significance of the
Michaelis-Menten parameters
• kcat : the catalytic constant
– Is a first order rate constant that refers to the
properties and reactions of the enzyme-substrate,
enzyme-intermediate, and enzyme-product
complexes.
– Often called the “turnover number”
The significance of the
Michaelis-Menten parameters
• KM : real and apparent equilibrium
constant
– Only KM = KdS for the simple M-M mechanism
– The KM is an apparent dissociation constant that may
be treated as the overall dissociation constant of all
enzyme-bound species
– In all cases is the substrate concentration at which
v = Vmax/2
4
The significance of the
Michaelis-Menten parameters
• kcat/KM : the specificity constant
– kcat/KM relates the reaction rate to the concentration of
free enzyme, rather than total enzyme.
– The kcat/KM is an apparent second-order rate constant
that refers to the properties and the reactions of the
free enzyme and free substrate
– v = [E] [S] kcat/KM
Mechanism of regulation of protein function
• Why?
– To ensure that the protein is only present in its active form in the
specific compartment where is needed
• How?
– Signal sequence
– Attachment of lipid tail that insert into membranes
– Structural interaction domain (e.g., recognize phosphorylation in
other protein)
• Localization is a dynamic process (e.g. transcription factors)
• When the protein is not in the location where it is needed, very often
it is maintained in an active conformation.
5
Protein activity can be regulated by
binding of an effector
• Binding of effector molecules can induce conformational changes
that produce inactive or active forms of the protein.
• Effectors may bind noncovalently or may modify the covalent
structure of the protein, reversibly or irreversibly.
• Often, a product of an enzyme reaction can act as a competitive
inhibitor.
• Ligands, including reaction products, may also bind to sites remote
from the active site (allosteric regulation).
S
P
conc. in cell 10 nM
X
P
Kd of allosteric regulator = 0.1 μM
active
P
inactive
Thermodynamic and kinetics of
disulfide bond formation in proteins
SH
P
S
+
GSSG
SH
+ 2 GSH
P
S
K = [P(SS)]eq [GSH]eq2 / [P(SH)2]eq [GSSG]eq Thermodynamics
Rate of oxidation = k [P(SH)2] [GSSG]
Kinetics
You have 2 different solutions containing:
a) 0.1 mM GSH and 1 mM GSSG
b) 1 mM GSH and 100 mM GSSG
How is the concentration of the oxidized and reduced protein at equilibrium
in each tube?
If you add the reduced protein to each tube, which one will have
the highest initial rate of oxidation ?
6