Enzyme Kinetics All diagrams and photos without attribution are MS Word clip art or the authors (RA Edwards) own work/photo.*Learning Objectives for Theme 2 Topic B Energy & Metabolic Pathways

3. Compare and contrast irreversible and reversible inhibition, and competitive and noncompetitive inhibition of enzymes.*

Whats Going On TodayReview the function of enzymes.

Find out how to measure the rate of an enzyme catalyzed reaction in a test tube.

Discover how the rate of a reaction depends on three different things: enzyme concentration [E], substrate concentration [S], and presence of an inhibitor. *

Review EnzymesReview of Enzyme function http://www.youtube.com/watch?v=E-_r3omrnxw&feature=relatedEnzymes vastly increase the rate of reaction by decreasing DG* (DE*, EA, G0,).Enzymes are quite specific and catalyze only one reaction.Nearly every reaction that occurs in living organisms is catalyzed by an enzyme.Many bio-reactions look like coupled reactions. Enzymes catalyze the two coupled reactions simultaneously.Often one of the coupled reactions is ATP + H2O ADP + PiTherefore: Enzymes look like energy coupling devices.*

CPRG(aq) + H2O(aq) CPR(aq) + galactoseLactaid, is an enzyme supplement (beta-glactosidase)

(CPRG = Chlorophenolred-galactoside)Example Reaction*

How to Measure How Fast Enzymes Work Add enzyme to substrate in buffer.When enzyme is added to substrate start a timer.After a shot time (1 or 2 minutes) measure the colour change by the absorbance of the product (CPR) at 572 nm.*

Calculating Rate in umol/min Use the absorbance to calculate the concentration of CPR.Use the conc. and the volume to calculate the amount (umoles) of CPR.Divide the number of umoles of product by the time (in minutes) to get the rate in umoles per minute.Example:A572 = 0.44 umoles of Product after 2.0 minutes. *Velocity = 0.44 umoles / 2.0 minutes = 0.22 umol/min.

Finding out how Rate Depends on [E] Use the same procedure as already described (add substrate, add enzyme, time to 1 minute and measure A572), but use several tubes. Use _________________________ enzyme in the different tube. Repeat to get duplicates and then repeat to get triplicate.Repeat previous calculation procedure & average triplicate rates. *

Plot Rate versus [E] *

Finding out how Rate Depends on [S] Use previous procedure, but add same amount of enzyme to several tubes which have different concentrations of substrate.Do step #1 in triplicate.Repeat previous calculation procedure & average triplicate rates. Substrate*

Plot Rate versus [S] Vmax 15 umol/min*

Km is the [S] needed to get Vmax Vmax 7.5 umol/minKm 4.0 uM*

How does Rate Depend on Inhibitor Use previous procedure, but add same amount of enzyme to same concentration of substrate to two tubes one has an inhibitor the other does not have the inhibitor.Do step #1 in triplicate on several substrate concentrations (with or without the same inhibitor concentration in pairs of tubes).Repeat previous calculation procedure & average triplicate rates. No Inhibitor *

Plot Rate with & without Competitive Inhibitor Vmax 15 umol/min is the same with and without inhibitor*

Competitive Inhibitor Inhibitor looks chemically somewhat like the substrate.Inhibitor binds at the active site in competition with the substrate.High concentrations of the substrate will out-compete the inhibitor.Active SiteE*

Noncompetitive Inhibition Inhibitor is not chemically similar to the substrate.Inhibitor binds at some other site on the enzyme (not the active site). High concentrations of the substrate do not out-compete the inhibitor.*

Reversible Inhibition Competitive Inhibition is one type of Reversible Inhibition because the inhibitor can be released from the enzyme.High [S] will knock the inhibitor off of the enzyme Vmax is unchangedNon-Competitive Inhibition is another type of Reversible Inhibition.High [S] will not knock the inhibitor from the E Vmax is decreased.*

Irreversible Inhibition Inhibitor reacts with the enzyme so that the enzyme doesnt catalyze properly.The substrate can NOT out-compete so that Vmax is decreased.*

5.1What is a common way to obtain the data needed to calculate the velocity of an enzyme catalyzed reaction?Measure the heat released by the reaction.Measure the equilibrium constant after the reaction has stopped.Measure the decrease in the substrate concentration.Measure the absorbance of the products.*

5.2How does the velocity (v) of an enzyme catalyzed reaction depend on [E] and [S]?

v increases linearly with both increasing [E] and increasing [S].v does not increase linearly with either [E] or [S].v increases linearly with increasing [E], but reaches a plateau with increasing [S].v increases reaches a plateau with increasing [E], increases linearly with increasing [S].*

***********Not same site**Chemically bound**Do not need to show these in class or go through them in class; are provided in lecture slides so that students will see them when download/print slides for class

*Do not need to show these in class or go through them in class; are provided in lecture slides so that students will see them when download/print slides for class