Download Quiz - Columbus Labs

Quiz
1
Common writing mistakes
• Avoid using general words such as “it”, “this”, and
“thing”. Use more informative words.
•Refer to Figures in text
•Provide Figure titles and legends to briefly describe the
figure and explain acronyms, color legend, and symbols.
•Try to avoid lists of facts; better to focus on explaining
one or two rather than leave the reader asking how or
why.
•Sentences should not be cluttered by long phrases that
can be reduced to one word
•Try to go back and rework text to be less conversational
and more like an essay
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Evaluation of Project 2
Understanding of scientific material (50 pts)
_______
Organization (20 pts)
_______
Grammar (10 pts)
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Use of additional references (10 pts)
_______
Creativity (5 pts)
_______
Strong introduction and conclusion (5 pts)
_______
Total (100 pts)
_______
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Readings for 10/30 and 11/1
Discussion about the discovery of the α-helix
and the β-sheet
discovery.pdf
Methods and Advanced Topics
proteomics_disease.pdf
Structure_drug.pdf
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What equations do I need to know?
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HH equation
MM equation (know the assumptions)
kcat
Catalytic efficiency
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Outline
• What Factors Influence Enzymatic Activity?
• What Are the General Features of Allosteric Regulation?
• Can a Simple Equilibrium Model Explain Allosteric
Kinetics?
• Is the Activity of Some Enzymes Controlled by Both
Allosteric Regulation and Covalent Modification?
– Thursday - Special Focus: Is There an Example in
Nature That Exemplifies the Relationship Between
Quaternary Structure and the Emergence of Allosteric
Properties? Hemoglobin and Myoglobin-Paradigms of
Protein Structure and Function
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What Factors Influence
Enzymatic Activity?
• Rate slows as product accumulates
• Rate depends on substrate availability
• Genetic controls - induction and repression
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Covalent modification
Proteolytic activation: zymogens
Multiple forms of enzymes: isozymes
Modulator proteins
Allosteric control
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Covalent Modification Can Regulate Enzyme Activity
Enzymes regulated by covalent modification are called interconvertible
enzymes.
The enzymes catalyzing the conversion of the interconvertible enzyme between
its two forms are called converter enzymes.
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Examples of regulation by
proteolytic cleavage
• Insulin
• Proteolytic enzymes of the digestive tract
• Blood Clotting
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Proinsulin/Insulin
Proinsulin is an 86-residue precursor to
insulin (the sequence shown here is human
proinsulin). Proteolytic removal of residues
31 to 65 yields insulin. Residues 1 through
30 (the B chain) remain linked to residues 66
through 87 (the A chain) by a pair of
interchain disulfide bridges.
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The proteolytic activation of chymotrypsinogen.
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The proteolytic activation of blood clotting
The cascade of
activation steps leading
to blood clotting. The
intrinsic and extrinsic
pathways converge at
Factor X, and the final
common pathway
involves the activation of
thrombin and its
conversion of fibrinogen
into fibrin, which
aggregates into ordered
filamentous arrays that
become cross-linked to
form the clot.
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Regulation of Enzymes – Isozymes
lactate dehydrogenase (LDH) example
NADH + H+
COOC=O
CH3
Pyruvate
NAD+
COOHO-C-H
Lactate
CH3
dehydrogenase
L-lactate
Active muscle tissue becomes anaerobic and produces pyruvate from glucose via glycolysis.
LDH regenerates NAD+ from NADH converting pyruvate to lactate so glycolysis can
continue. The lactate produced is released into the blood.
The muscle LDH isozyme (A4) works best in the NAD+-regenerating direction. Heart tissue is
aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate
to fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited by 13
excess pyruvate so the energy won’t be wasted.
Modulator Proteins: An example
PKA – cAMP dependent protein kinase
The two R (regulatory) subunits bind cAMP (KD = 3 x 10-8 M); cAMP binding releases the
R subunits from the C (catalytic) subunits. C subunits are enzymatically active as
monomers.
PKA phosphorylates protein sequence
R(R/K)X(S/T)
R subunit has psuedosubstrate (red) with sequence
RRGAI where Ala stericly mimics the Ser, but can not
be phosphorylated.
cAMP induces a conformational change that releases
the R subunit freeing the active site so that PKA can
phosphorylate targets.
This complex also includes ATP (yellow) and two Mn2+
ions (violet) bound at the active site
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Allosteric Regulation
• allosteric means other site and an 'allosteric enzyme' is
one with two binding sites - one for the substrate and
one for the allosteric modifier molecule, which is not
changed by the enzyme so it is not a substrate.
• The molecule binding at the allosteric site is not called
an inhibitor because it does not necessarily have to
cause inhibition - so they are called modifiers.
• A negative allosteric modifier will cause the enzyme to
have less activity
• a positive allosteric modifier will cause the enzyme to be
more active.
• allosteric regulation requires the enzyme be multimeric
(ie. a dimer, trimer, tetramer etc.).
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General Features of Allosteric Regulation
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Action at "another site"
Enzymes situated at key steps in metabolic pathways are modulated
by allosteric effectors
These effectors are usually produced elsewhere in the pathway
Effectors may be feed-forward activators or feedback inhibitors
Kinetics are sigmoid ("S-shaped")
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Monod, Wyman, Changeux (MWC) Model of
Allosteric Regulation
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Monod, Wyman, Changeux (MWC)
Model: allosteric proteins can exist in two
states: R (relaxed) and T (taut)
In this model, all the subunits of an
oligomer must be in the same state
T state predominates in the absence of
substrate S
S binds much tighter to R than to T
Cooperativity is achieved because S
binding increases the population of R,
which increases the sites available to S
Ligands such as S are positive
homotropic effectors
Molecules that influence the binding of
something other than themselves are
heterotropic effectors (F)
KR
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The Monod - Wyman - Changeux model – “K” system
Graphs of allosteric effects for a tetramer (n = 4) in terms of Y, the saturation function,
versus [S]. Y is defined as [ligand-binding sites that are occupied by ligand]/[ total ligandbinding sites]. c = KR/KT. (substrate dissociation constant for the two states; when c = 0, KT
is infinite, T doesn’t bind to S)
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MWC Heterotropic allosteric effects: “K”
The linked equilibria lead to changes in the relative amounts of R and T and, therefore,
shifts in the substrate saturation curve. This behavior, depicted by the graph, defines an
allosteric “K” system. The parameters of such a system are: (1) S and A (or I) have
different affinities for R and T and (2) A (or I) modifies the apparent K0.5 for S by shifting
the relative R versus T population.
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allosteric effects: “V”
In the “K” model – K0.5 (relative KM)
changes and Vmax is constant
In the “V” model – Vmax changes and
K0.5 is constant
The positive heterotropic effector (A)
increases Vmax and the negative
heterotropic effector (I) decreases in
Vmax
If R and T have the same affinity for the substrate, but
differ in catalytic activity and their affinities for A and I the
the “V” model situation arises
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“K” vs “V” MCW model
• “K” is more likely when [S] is the limiting
• “V” is more likely when [S] is saturating
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The Koshland-Nemethy-Filmer sequential model for allosteric regulation
(a) S-binding can, by induced fit, cause a
conformational change in the subunit to
which it binds.
(b) If subunit interactions are tightly coupled,
binding of S to one subunit may cause
the other subunit to assume a
conformation having a greater (positive
homotropic) or lesser (negative
homotropic) affinity for S. That is, the
ligand-induced conformational change in
one subunit can affect the adjoining
subunit. Such effects could be
transmitted between neighboring peptide
domains by changing alignments of
nonbonded amino acid residues.
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The Koshland-Nemethy-Filmer sequential model for allosteric regulation
Theoretical curves for the
binding of a ligand to a
protein having four
identical subunits, each
with one binding site for
the ligand. The fraction
of maximal binding is
plotted as a function of
[S]/K0.5.
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Examples of protein regulation
where multiple mechanisms are used
glycogen phosphorylase
Allosteric Regulation and Covalent Modification
• GP cleaves glucose units from nonreducing ends of
glycogen
• A phosphorolysis reaction
• Muscle GP is a dimer of identical subunits, each with PLP
covalently linked
• There is an allosteric effector site at the subunit interface
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The glycogen phosphorylase reaction.
The phosphoglucomutase reaction
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Structure of a glycogen phosphorylase
The structure of a glycogen phosphorylase monomer, showing the locations of
the catalytic site, the PLP cofactor site, the allosteric effector site, the glycogen
storage site, the tower helix (residues 262 through 278), and the subunit
interface.
Glycogen phosphorylase dimer.
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Glycogen Phosphorylase
Allosteric Regulation and Covalent Modification
• Pi is a positive homotropic effector (a)
• ATP is a negative heterotropic effector (feedback inhibitor) (b)
• Glucose-6-P is a negative heterotropic effector (i.e., an
inhibitor)
• AMP is a positive heterotrophic effector (i.e., an activator) (c)
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Conformational Change of glycogen phosphorylase upon phosphorylation
In this diagram of the glycogen phosphorylase
dimer, the phosphorylation site (Ser14) and the
allosteric (AMP) site face the viewer.
Access to the catalytic site is from the opposite
side of the protein.
The diagram shows the major conformational
change that occurs in the N-terminal residues
upon phosphorylation of Ser14. The solid black
line shows the conformation of residues 10 to
23 in the b, or unphosphorylated, form of
glycogen phosphorylase. The conformational
change in the location of residues 10 to 23
upon phosphorylation of Ser14 to give the a
(phosphorylated) form of glycogen
phosphorylase is shown in yellow.
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Regulation of GP by Covalent
Modification
• In 1956, Edwin Krebs and
Edmond Fischer showed that
a ‘converting enzyme’ could
convert phosphorylase b to
phosphorylase a
• Three years later, Krebs and
Fischer show that this
conversion involves covalent
phosphorylation
• This phosphorylation is
mediated by an enzyme
cascade
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The hormone-activated enzymatic cascade that leads to
activation of glycogen phosphorylase.
Cyclic AMP is the intracellular agent of extracellular
hormones - thus a ‘second messenger’
Hormone binding stimulates a GTP-binding protein (G
protein), releasing Gα(GTP)
Binding of Gα(GTP) stimulates adenylyl cyclase to make
cAMP
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The adenylyl cyclase reaction
The adenylyl cyclase reaction yields 3',5' -cyclic AMP and pyrophosphate. The
reaction is driven forward by subsequent hydrolysis of pyrophosphate by the
enzyme inorganic pyrophosphatase.
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G-protein signal transduction cascade
Hormone (H) binding to its receptor (R) creates a hormone;receptor complex (H:R) that
catalyzes GDP-GTP exchange on the α -subunit of the heterotrimer G protein (Gαβγ ),
replacing GDP with GTP. The Gα -subunit with GTP bound dissociates from the βγ -subunits
and binds to adenylyl cyclase (AC). AC becomes active upon association with Gα :GTP and
catalyzes the formation of cAMP from ATP. With time, the intrinsic GTPase activity of the Gα subunit hydrolyzes the bound GTP, forming GDP; this leads to dissociation of Gα :GDP from
AC, reassociation of Gα with the βγ subunits, and cessation of AC activity. AC and the
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hormone receptor H are integral plasma membrane proteins; Gα and Gβγ are membraneanchored proteins.