1a' Draw the individual titration curves for His119 and His12 in ribonuclease

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1a. Draw the individual titration curves for
His119 and His12 in ribonuclease if Asp121 is
mutated to alanine. Combine the titration curves
and show an activity profile (activity vs. pH).
His 119
OR
pKa
-O - P O

N - H
HN
O
Asp121
His119
activity
His119 with D121A
pH
His
HisH
For His119, mutation of Asp121 to Ala will
destabilize HisH and lower the pKa. The curve
will shift to the left.
Ka H His / HHis
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1a. Draw the individual titration curves for
His119 and His12 in ribonuclease if Asp121 is
mutated to alanine. Combine the titration curves
and show an activity profile (activity vs. pH).
His12 with D121A
His12
activity
pH
His
HisH
For His12, mutation of Asp121 to Ala will have
little or no effect.
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Combining the individual titration curves yields
an activity profile.
Ribonuclease
optimum pH
activity
Ribonuclease with D121A mutation
pH
His
HisH
Net effect of mutation is to reduce the pH range
where the enzyme is active and to shift the pH
optimum to lower pH
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1b. Draw the individual titration curves for
His119 and His12 in ribonuclease if Ala40 near
His12 is mutated to lysine. Combine the
titration curves and show an activity profile
(activity vs. pH).
pKa
His119
activity
His119 with A40K
pH
His
HisH
For His119, mutation of Ala40 to lysine will have
little or no effect.
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1b. Draw the individual titration curves for
His119 and His12 in ribonuclease if Ala40 near
His12 is mutated to lysine. Combine the
titration curves and show an activity profile
(activity vs. pH).
Ala40-Lys41
His 12
His12
His12 with A40K
activity
O - H
NH
N
pH
His
HisH
For His12, mutation of Ala40 to lysine will
destabilize HisH and shift equilibrium toward
neutral His. This will shift the titration curve
to the left.
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Combining the individual titration curves yields
an activity profile.
His12
His12 with A40K
activity
His119 (no change)
pH
His
HisH
Net result will be to broaden the pH range where
the enzyme is active, and to shift the pH optimum
to lower pH
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Catalytic Mechanisms and Strategies

• Lecture 4 How is molecular motion involved in
  catalysis?
• Vibrational motions vs. large scale motions
• Diffusion
• Hinging and shear motions in catalysis

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Substrate
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Enzyme in the closed state without substrate
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Protein Dynamics - T4 Lysozyme
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Protein Dynamics
Atomic fluctuations. Vibrations. 10-15 to
10-12 s lt1Å Group motions. (covalently
linked units) 10-12 10-3 s lt 1 Å 50
Å Rotation of methyl groups. 10-12 10-9
s Flips of aromatic rings. 10-9 10-6
s Domain motions. 10-8 10-3 s Proline
isomerization. gt 10-3 s
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Vibrations stretches bends torsions Rotation
s Translations
u vibrational frequency 1/2? (?/?)1/2
? reduced mass m1m2/m1 m2
K vibrational force constant
The larger the mass, the lower the frequency
MD
?r rotational correlation time 3 V
?solvent / kT
The larger the volume or more viscous the
solution, the slower the rotation
D diffusion coeff. kT / f f frictional
coeff. 6??solvent r x2 2Dt
The larger the frictional coeff., slower the
translation.
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Catalytic Mechanisms and Strategies

• Lecture 5 How is molecular motion involved in
  catalysis?
• Vibrational motions vs. large scale motions
• Diffusion
• Hinging and shear motions in catalysis

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Diffusion
D diffusion coeff. kT / f f frictional
coeff. 6??solvent r x2 2Dt kdiffusion 4?N
(DP DL) rPL rPL distance for binding to
occur Dp,l diffusion coefficient of protein or
ligand kdiffusion 109 M-1 s-1
1011 M-1 s-1 with favorable
electrostatics
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using x2 2Dt
104
103
Average distance moved in Å
10-6 cm2/s
10-7 cm2/s
5-15 x 10-7 cm2/s Typical range of translational
diffusion coefficients for proteins
100
10-8 cm2/s
10
10-10
10-8
10-6
10-4
10-2
1
Time (s)
Creighton, Figure 7.3
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using x2 2Dt
104
103
Average distance moved in Å
10-6 cm2/s
10-7 cm2/s
5-15 x 10-7 cm2/s Typical range of translational
diffusion coefficients for proteins
100
10-8 cm2/s
10
10-10
10-8
10-6
10-4
10-2
1
Time (s)
Creighton, Figure 7.3
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Enzymes for which kcat/Km is close to diffusion
controlled association rate
Enzyme substrate kcat Km kcat/Km
(M-1 s-1 ) Acetylcholine esterase acetylcholine
1.4 x 104 9 x 10-5 1.6 x 108 Carbonic
anhydrase CO2 1 x 106 0.012 8.3 x
107 Triosphosphate G-3-P 4.3 x
103 5 x 10-4 2.4 x 108 isomerase
Fersht, Table 4.5
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Catalytic Mechanisms and Strategies

• Lecture 5 How is molecular motion involved in
  catalysis?
• Vibrational motions vs. large scale motions
• Diffusion
• Hinging and shear motions in catalysis

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I. Motions of Fragments Smaller than Domains
What is a domain? A. Motion is predominantly
shear Proteins for which two or more
conformations are known Dihydrofolate Reductase
(DHFR) Insulin Thymidylate Synthase
Bacteriorhodopsin (bR)
Mark Gerstein, Yale University http//bioinfo.mbb.
yale.edu/MolMovDB
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Bacteriorhodopsin
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Bacteriorhodopsin
The major conformational changes in the
photocycle involve small shifts in the position
of two of the helices (F and G) relative to the
others. Thus, the small sliding motions in
bacteriorhodopsin, as well as that in the
aspartate receptor, appear to be consistent with
a shear mechanism.
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B. Motion is predominantly hinge Proteins for
which two or more conformations are
known Annexin V (Trp motion) Cystatin Enolase
Foot and mouth disease virus HIV-1 protease
Hhal Methyltransferase Immunoglobulin (CDR
motion) Isocitrate Dehydrogenase (IDH) Lactate
Dehydrogenase (LDH) Lipase Malate Dehydrogenase
(MDH) Seryl-tRNA synthetase Triglyceride Lipase
Triose Phosphate Isomerase (TIM) Yersinia
Protein Tyrosine Phosphatase ras Protein
recvinm
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Triosphosphate isomerase
DHAP G3P
Loop closure with 2 hinges moves CA atoms 7 A.
When the enzyme binds its substrate the loop
closes over the active site, shielding the
substrate from water. The loop appears to close
as a rigid lid, stabilized by internal hydrogen
bonds. The closure involves the filling of a
cavity near the base of the helix to which the
loop is connected and the formation of new
hydrogen bonds and contacts.
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HIV Protease Monomer
Two large loop regions, that together comprise
one quarter of the structure, move CA atoms 7 Å
Location of catalytic Asp
Substrate binding pocket
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II. Domain Motions A. Motion is predominantly
shear Proteins for which two or more
conformations are known Alcohol Dehydrogenase
(ADH) Aspartate Amino Transferase (AAT) Citrate
Synthase Endothiapepsin Glyceraldehyde-3-phospha
te Dehydrogenase (GAPDH) Glycerol Kinase
Hexokinase Human Interleukin 5
Phosphofructokinase (PFK) (not allosteric
transition) Trp Repressor (TrpR)
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Hexokinase
with glucose bound
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Description of shear motion in hexokinase
Shear motion with XBAaba layering and prominent
crossed helices at interdomain interface
Hexokinase has 3 moving layers in one domain that
shift relative to 3 rigid layers in the other
domain. This distinctive layering pattern is of
the form XBAabx, where abx are the 3 moving
layers and XBA the rigid layers. The interface
between the two middle layers is where the major
shear motion occurs. One layer of helices from
the mobile domain (a) slides over a layer of
helices from the motionless domain (A). Helices
in these two layers, which in a sense form gears
upon which the domains slide, are often
'crossed'. Near to the a and A layer helices, the
ligand binds in the interdomain cleft . Packed
onto either side of the central layers of helices
(a and A) are sheets (b and B) from the mobile
and motionless domains, respectively. The mobile
sheet (b) forms a second moving layer, which
slides over the helices, and packed onto the
other face of this sheet is a third layer (x)
which moves with the sheet. Symmetrical to this
third moving layer (x), a third motionless layer
(X) is packed onto one side of the static sheet
(B). In hexokinase layer x and X are made up of
helices.
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B. Motion is predominantly hinge Proteins for
which two or more conformations are
known Acetylcholinesterase Adenylate Kinase
(ADK) Annexin V (breathing motion) Calbindin
Calmodulin Canine Lymphoma Immunoglobulin
(Fc-Fab hinge) Catabolite Gene Activator Protein
(CAP) Cell Adhesion Molecule CD2 DNA Polymerase
Beta (Pol Beta) Diphtheria Toxin (DT) E. coli.
Periplasmic Dipeptide Binding Protein Family-5
Endoglucanase CelC Formate Dehydrogenase (FDH)
Glutamate Dehydrogenase (GDH) Glutamine Binding
Protein
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B. Motion is predominantly hinge (contd)
Proteins for which two or more conformations are
known GroEL domain Heat Shock Transcription
Factor Interferon-gamma Iron Sulfur Protein
bc1 complex Lactoferrin Lysine/Arginine/Ornithine
(LAO) binding protein Maltodextrin Binding
Protein (MBP) Phosphoglycerate Kinase
Recoverin T4 lysozyme mutants Ile3-gtPro
Met6-gtIle Tomato Bushy Stunt Virus (TBSV) Coat
Protein Troponin-C Tryptophan Synthase c-Src
tyrosine kinase cAMP-dependent Protein Kinase
(catalytic domain)
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Glutamate binding protein
Classic hinging motion of two domains.
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C. Motion involves partial refolding of tertiary
structure Proteins for which two or more
conformations are known Ga HIV-1 Reverse
Transcriptase Haemagglutinin Serpins
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III. Larger Movements than Domain Movements
involving the Motion of Subunits A. Motion
involves an allosteric transition Proteins for
which two or more conformations are
known Aspartate Transcarbamoylase (ATCase)
Fructose-1,6-biphosphatase Glycogen
Phosphorylase (GP) Hemoglobin (Hb) Lac
Repressor Core (Allosteric Motion) Lac Repressor
upon binding DNA (Subunit motion via
Tetramerization domain) Phosphofructokinase
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B. Motion does not involve an allosteric
transition Proteins for which two or more
conformations are known Ca pump/ATPase Aspartat
e Receptor Bam HI Endonuclease Immunoglobulin
(VL-VH movement) S. cerevisiae PPR1 Zn-finger
DNA recognition protein. Erythopoietin Receptor
F1-ATPase Polymerase Processivity Factor PCNA
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ATP synthase
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Energy transduction in cells is driven by proton
gradients across cell membranes
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