Lab: principles of protein purification

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Enzyme Kinetics C483 Spring 2013.

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Presentation transcript:

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?

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?

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.

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?

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”

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’

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

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

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

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

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

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 = ?