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 Utahgoldenberg@biology.utah.edu

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

Direction Change

Warning!

Back to Proteases

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.

Clicker Question #3

What bond does a protease cleave?

H

N

O

O

N

H

CH 3

O

NH 3

+

A B

C

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)!

Image Formation with a Lens

Duck Duck Image

Lens

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.

Diffraction from a Duck

Film

A Real Diffraction Pattern From a Pretend Duck

Taylor, C. & Lipson, H. (1964). Optical Transforms: Their preparation and application to X-ray diffractionproblems. Cornell Univ. Press, Ithaca, NY.