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Biological Chemistry LaboratoryBiology 3515/Chemistry 3515
Spring 2020
Lecture 9
Quiz Questions andIntroduction to Proteases and Protein Structure
4 February 2020c©David P. Goldenberg
University of [email protected]
First Quiz: Thursday, 6 February
Review session:
Wednesday, 5 February, ASB 210
What to bring to the review session:
Questions!
What to bring to the quiz:
• A sharp pencil (much preferred over pen)
• A good eraser (just in case)
• A scientific calculator
• Your wits
A Quiz Question from 2018
The stated problem:
• A buffer solution is prepared by mixing:
0.2 moles MES, a weak acid with a pKa of 6.21 L of water150 mL of 1 M NaOH
• What is the expected pH?
Some things to consider:
• What is this question about?
• What will the answer look like?
• What will determine the answer?
Clicker Question #1
HA −−−⇀↽−−− A– + H+, pKa= 6.2
What happens when HA is dissolved in water?
(Choose 1)
A) Nearly all of the HA dissociates into A– and H+.
B) Half of the HA dissociates into A– and H+.
C) A small amount of the HA dissociates into A– and H+
D) None of the HA dissociates into A– and H+.
Clicker Question #2
HA −−−⇀↽−−− A– + H+, pKa= 6.2
Add 150 mL of 1 M NaOH to 0.2 moles HA
What will the pH be, approximately?
A) pH < 5
B) 5 ≤ pH < 6.2
C) pH = 6.2
D) 6.2 < pH ≤ 7.5
E) pH > 7.5
Calculating the pH
HA −−−⇀↽−−− A– + H+, pKa= 6.2
pH − pKa = log[A−]
[HA]= log
moles A–
moles HA
The total number of moles HA and A– is 0.2.
0.15 moles of HA are converted to A–
moles HA = 0.05, moles A– = 0.15
pH = pKa + log0.150.05
= 6.2 + 0.5
= 6.7
General Protease Mechanism is Nucleophilic Substitution
H2NC
C
C
O
NuC
C
O
NH
C
Nu
+C
C
O-
NH
C
Nu
HA
+
Water Can Act as the Nucleophile,
but Must be Activated by a Base
H2NCα
Cα
C
O
OHCα
C
O
NH
Cα
+Cα
C
O-
NH
Cα
HA
H
O
H
H
O-
H
O
B
BH
Why is this reaction so slow in the absence of an enzyme?
How do enzymes enhance the rate?
Carboxyl Groups Activate H2O in Aspartyl Proteases
Asp Asp Asp Asp
O
N H
O H H
O –
N H
HO
O
– O H +
O –
O
O
O
OH
HO
Examples include pepsin and HIV protease
Serine Proteases Employ a Two-Step Mechanism
Enzyme-Substrate Complex Acyl Intermediate Enzyme + Products
O
O
O H
H
OH
O
N H
+ H 3 N
OH
O
O –
+ H 3 N
In step 1, a serine hydroxyl is the nucleophile.
In step 2, a water molecule is the nucleophile.
Both steps require activation by a base.
Examples include trypsin, chymotrypsin, blood clotting factors and manyothers.
How Do We Know What We Know About Serine Proteases?
Information
Observations
Experiments
Data
Knowledge
Organized information
Theories
Predictions
Chemical and biochemical data:
• Enzyme kinetics
• Studies with inhibitors
• Chemical analysis, e.g., active site labeling
Structural analysis, mostly X-ray crystallography
Clicker Question #4: How Big is an Enzyme?
A) 10−10m
B) 10−9m
C) 10−8m
D) 10−7m
E) 10−6m?
No wrong answers (for now)!
Optical Magnification
Duck
Magnified Duck Image
Lens
A B
As the object is brought closer to the lens:
Image moves further from the lens and becomes larger.
Magnification = B/A
Magnification, in principle, is not limited, but resolution is.
Imaging With a Lens - a Wave Interpretation
Image is formed at points where waves are brought back in phase.
Points in the object must be separated by at least ∼ 1/2 wavelength to giverise to separate points in the image.
The Electromagnetic Spectrum
Illustration From: McMurry, J. & Fay, R. (2004). Chemistry . Prentice-Hall, 4th edition.
Why Not an X-Ray Microscope?
Scattering from individual atoms is very weak, especially from elementswith low atomic numbers.
Very difficult to make lenses for X-rays.
In crystallography:
• Use crystals to increase the total scattering intensity.
• Use a mathematical technique, the Fourier transform, to do the job of a lens.