1. Lab: principles of protein purification
Chapter 4: Protein structure 2°,3°,4°
Principles driving folding, formation of particular 3D structures
Common 2° structures
Principles. How do they differ? Why do they form?
Protein folding process
What do we know? What did Anfinsen’s experiment tell us?

2. Chapter 5: Protein function, esp. principles of reversible binding
What is Kd? How does it relate to affinity? (What is “affinity” for that matter?)
What is q?
Binding of myoglobin to O2
Binding of hemoglobin to O2
Cooperativity: why biologically? More important: why structurally?

3. Enzyme kinetics Rate of enzyme catalyzed reaction
How the rate changes in response to experimental parameters
eg. initial [substrate], presence of inhibitor, pH, temperature, etc.

4. Why enzyme kinetics? Biology Chemistry Pharmacology
Preferred substrate? In which pathway is the enzyme involved?
Mechanisms of regulation?
Chemistry
Mechanism of catalysis?
Pharmacology
How an inhibitor works?
How an inhibitor might be improved?

5. General reaction rates
Reaction rate depends on concentration of substrate(s)
Note: many times water is a substrate
~constant concentration, ~no effect on rate
Rate = k [A]x
A → B + C
Reaction order
How does the rate depend
on concentration?
Rate doubles as [A] doubles?
First order reaction (x = 1)
Proportionality constant
“Rate constant”

6. Enzyme kinetics Monitor rate of enzyme catalyzed reaction
Change substrate concentration, pH, temp…
Curve for rate (V) vs. [S] of first order, uncatalyzed reaction?
Many enzymes, V vs. [S] curve is hyperbolic
Vmax
Enzyme is ‘saturated’ with substrate
‘Michaelis-Menten enzymes’

7. Michaelis-Menten kinetics
Assumptions
Binding of E and S is reversible
Product formation (k2 step) is rate-limiting
Rate is dependent upon conc of ES
Steady state assumption
Monitor ‘steady state’ where ES is constant
k1 step is very fast
[S] >> [E]
ES forms as quickly as it breaks down
Initial velocity assumption
Ignore k-2

8. Michaelis-Menten kinetics
Assumption 2: k2 is rate-limiting
ie. k2 and [ES] define the rate
Vo = k2[ES]
Assumption 3: Rate of [ES]formation = rate of [ES]breakdown
k1[E]free[S] = (k-1 + k2)[ES]
From these assumptions, you can derive the:
Michaelis-Menten equation

9. Michaelis-Menten Values
The Michaelis Constant:
Vmax
Rate of reaction at infinite substrate concentration

10. Michaelis-Menten kinetics
Michaelis-Menten equation
Km = [S] at which reaction rate is ½ Vmax
Michaelis curve

11. Michaelis-Menten kinetics
‘Linearize’ substrate saturation curve
Lineweaver-Burk plot
Reciprocal of both sides of MM equation
Y-intercept= ?
X-intercept= ?
Small errors at low [S] have major effects

12. Michaelis-Menten kinetics
‘Linearize’ substrate saturation curve
Eadie Hofstee plot
Multiply both sides of MM equation by Km + S
What do you plot for linearity?
Y-intercept = ?
Slope = ?