6 Enzymes II W. H. Freeman and Company

Transcription

1 6 Enzymes II 2017 W. H. Freeman and Company

2 The role of an enzyme in an enzyme-catalyzed reaction is to: A. bind a transition state intermediate, such that it cannot be converted back to substrate. B. ensure that all of the substrate is converted to product. C. ensure that the product is more stable than the substrate. D. increase the rate at which substrate is converted into product E. make the free-energy change for the reaction more favorable. iclicker 1

3 The role of an enzyme in an enzyme-catalyzed reaction is to: A. bind a transition state intermediate, such that it cannot be converted back to substrate. B. ensure that all of the substrate is converted to product. C. ensure that the product is more stable than the substrate. D. increase the rate at which substrate is converted into product E. make the free-energy change for the reaction more favorable. iclicker 1

4 Three parameters to characterize kinetics of enzyme-catalyzed reaction: Vmax, maximum velocity Km, the Michaelis constant Kcat, turnover number

5 Vmax is reached when the enzyme is saturated with substrate Vmax depends on the amount of enzyme. Kcat = Vmax / [Enzyme] Kcat is a turnover number Kcat measures velocity independent of enzyme concentration. Kcat is a constant for an enzyme under given condition. It is a number of substrate molecules converted to product in a given unit of time on a single enzyme molecule.

6 Km is a constant for the enzyme (Michaelis constant). It is a substrate concentration required to get Vmax /2. Km is a measure of enzyme s affinity for it s substrate. Km is inversely related to affinity (the higher Km, the lower affinity of the enzyme to it s substrate; the lower Km, the higher affinity)

7 Leonor Michaelis and Maud Menten derived an equation to describe the initial reaction velocity as a function of substrate concentration. When V o = ½ V max, K M =[S].

8 Lineweaver-Burk Plot: Linearized, Double-Reciprocal Km is a negative reciprocal of the X- intercept Vmax is an inverse of Y intercept

9 What units does Km have? A. M/sec B. Concentration (M, for example) C. % D. Sec E. 1/sec iclicker 2

10 What units does Km have? A. M/sec B. Concentration (M, for example) C. % D. Sec E. 1/sec iclicker 2

11 Enzyme Inhibition Inhibitors are compounds that decrease an enzyme s activity. Irreversible inhibitors (inactivators) react with the enzyme. One inhibitor molecule can permanently shut off one enzyme molecule. They are often powerful toxins but also may be used as drugs. Reversible inhibitors bind to and can dissociate from the enzyme. They are often structural analogs of substrates or products. They are often used as drugs to slow down a specific enzyme. Reversible inhibitor can bind to: the free enzyme and prevent the binding of the substrate. the enzyme-substrate complex and prevent the reaction.

12 Reversible Inhibitors Inhibitor resembles regular substrate; it binds at the active site Inhibitor binds to the enzymesubstrate complex Inhibitor binds to the regulatory site, not to the active site; it binds enzyme with or without substrate

13 Competitive Inhibition Competes with substrate for binding binds active site does not affect catalysis Inhibition can be overcome by the amount of substrate No change in V max ; K M Lineweaver-Burk: lines intersect at the y-axis.

14 Competitive Inhibition No change in V max ; K M Inhibition can be overcome by the amount of substrate

15 Uncompetitive Inhibition Only binds to ES complex does not affect substrate binding inhibits catalytic function V max ; K M No change in ratio K M /V max Lineweaver-Burk: lines are parallel.

16 Uncompetitive Inhibition Vmax ; KM ; No change in ratio KM / Vmax

17 Noncompetitive Inhibition Inhibitor has nothing in common with the substrate. It binds to enzyme at the regulatory site different from active site Noncompetitive inhibitor decreases the overall number of active enzyme molecules that are bound to substrate. Decrease in V max ; no change in K M

18 Noncompetitive Inhibition Vmax ; KM is unchanged

19 There are 4 graphs below corresponding 4 conditions: no inhibition; competitive inhibition; noncompetitive inhibition; uncompetitive inhibition Which line corresponds to competitive inhibition condition? A.1 B.2 C.3 D.4 E.None of the above iclicker 3

20 There are 4 graphs below corresponding 4 conditions: no inhibition; competitive inhibition; noncompetitive inhibition; uncompetitive inhibition Which line corresponds to competitive inhibition condition? A.1 B.2 C.3 D.4 E.None of the above iclicker 3

21 How cells control enzymes 1. Allosteric regulation (binding of non-substrate molecule) 2. Covalent modification 3. Access to substrate 4. Control of enzyme synthesis/ breakdown Combinations of methods can be used

22 Allosteric Regulators Allosteric effectors or modulators are generally small chemicals. Allosteric effectors can be positive (improve enzymatic catalysis) negative (reduce enzymatic catalysis).

23 Feedback Inhibition Allosteric enzymes are regulated by products of the pathways under their control The conversion of A to B is the committed step, because once this occurs, B is committed to being converted into F. Allosteric enzymes catalyze the committed step of metabolic pathways. The pathway product F inhibits enzyme e 1 by binding to a regulatory site on the enzyme that is distinct from the active site.

24 Allosteric Regulators The kinetics of allosteric regulators differ from Michaelis-Menten kinetics. The reaction velocity of allosteric enzymes displays a sigmoidal relationship to substrate concentration.

25 Allosteric Regulators The kinetics of allosteric regulators differ from Michaelis-Menten kinetics.

26 The effect of regulators on the allosteric enzyme aspartate transcarbamoylase (ATCase) ATCase catalyzes an early reaction in the synthesis of pyrimidine nucleotides, where aspartate is a substrate CTP is a the end-product of the pathway; it is a feedback inhibitor of the enzyme ATP is an allosteric activator

27 What type of inhibition can be overcome by increasing amount of substrate? A. Competitive B. Noncompetitive C. Uncompetitive D. All of the above E. None of the above iclicker 3

28 What type of inhibition can be overcome by increasing amount of substrate? A. Competitive B. Noncompetitive C. Uncompetitive D. All of the above E. None of the above iclicker 3

29 What type of inhibition can be reversed? A. Competitive B. Noncompetitive C. Uncompetitive D. All of the above E. None of the above iclicker 4

30 What type of inhibition can be reversed? A. Competitive B. Noncompetitive C. Uncompetitive D. All of the above E. None of the above iclicker 4