Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental...
-
Upload
baldric-wilson -
Category
Documents
-
view
229 -
download
1
Transcript of Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental...
![Page 1: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/1.jpg)
Enzyme Kinetics
Chapter 6
![Page 2: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/2.jpg)
Kinetics
• Study of rxn rates, changes with changes in experimental conditions
• Simplest rxn: S P
– Rate meas’d by V = velocity (M/sec)
– Depends on k, [S]
![Page 3: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/3.jpg)
Michaelis-Menten Kinetics• Gen’l theory rxn rate w/
enzymatic catalysis
• Add E, ES to rxn:
E + S ES E + P
• Assume little reverse rxn E + P ES
So E + S ES E + P
• Assign rate constants k1, k-1, k2
![Page 4: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/4.jpg)
• Assume: Vo condition -- [S] >>> [E]
– Since S used up during rxn, can’t be limiting
• Assume: All E goes to ES
• Assume: Fixed amt enzyme
– If all E ES, will see max rate of P formed
– At steady state
rate form’n ES = rate breakdown ES
![Page 5: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/5.jpg)
Exper’l Findings:
– As incr [S], V incr’s linearly up to some max V
– At max V, little V incr regardless of [S] added
![Page 6: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/6.jpg)
![Page 7: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/7.jpg)
M-M Relates [E], [S], [P] Exper’ly Provable Variables
• New constant:
KM = (k2 + k-1) / k1
• M-M eq’n:
Vo = (Vmax [S]) / (KM + [S])
![Page 8: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/8.jpg)
• Quantitative relationship between
– Initial velocity
– Max rate of rxn
– Initial [S]
![Page 9: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/9.jpg)
Exper’l Definition of KM
• At ½ Vmax (substitute ½ Vmax for Vo)
• Divide by Vmax
• Solve for KM
• KM = [S]
• So when Vo = ½ Vmax , KM = [S]
![Page 10: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/10.jpg)
![Page 11: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/11.jpg)
Difficult to Determine Variables from M-M Plot
• Hard to measure small changes in V
• Use double reciprocal plot straight line
• Lineweaver-Burk (Box 6-1)
![Page 12: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/12.jpg)
![Page 13: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/13.jpg)
KM
• [S] at which ½ enz active sites filled
• Related to rate constants
• In living cells, value close to [S] for that E
– Commonly enz active sites NOT saturated w/ S
![Page 14: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/14.jpg)
• May describe affinity of E for S ONLY if k-1 >>> k2
– Right half of rxn equation negligible
– KM = k-1 / k1
– Describes rate form’n, breakdown of ES
• Considered dissociation constant of ES complex
– Here, KM value indicates strength of binding E-S
– In real life, system is more complex
![Page 15: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/15.jpg)
![Page 16: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/16.jpg)
Other Kinetics Variables
• Turnover #
– kcat
– # S molecules converted P by 1 enz molecule per unit time
– Use when enz is fully sat’d w/ S
![Page 17: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/17.jpg)
![Page 18: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/18.jpg)
Comparisons of Catalytic Abilities• Optimum KM, kcat values for each E
• Use ratio to compare catalytic efficiencies
• Max efficiency at kcat / KM = 107– 108 M-1 sec-1
– Velocity limited by E encounters w/ S
– Called Diffusion Controlled Limit
![Page 19: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/19.jpg)
![Page 20: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/20.jpg)
Kinetics When>1 Substrate• Random order = E can accept
either S1 or S2 first
• Ordered mechanism = E must accept S1 first, before S2 can bind
• Double displacement (or ping-pong) = S1 must bind and P1 must be released before S2 can bind and P2 is released
![Page 21: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/21.jpg)
![Page 22: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/22.jpg)
Inhibition• Used by cell to control catalysis
in metabolic pathways
• Drugs, toxins alter catalysis by inhib’n
• Used as tools to study mechanisms
• Irreversible
• Reversible
– Includes competitive, noncompetitive, uncompetitive
![Page 23: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/23.jpg)
Irreversible Inhibition
• Inhibitor binds tightly to enz
• Dissociates slowly or not at all
• Book example: DIFP
• Includes suicide substrate inhibitors
![Page 24: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/24.jpg)
![Page 25: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/25.jpg)
Reversible Inhibition
• Inhibitor may bind at active site or some distal site
• Binding reversible
• Temporarily inhibits E, S binding or proper rxn
• Can calculate KI
![Page 26: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/26.jpg)
• Competitive
– “Appear as S”
– Bind active site
•So compete w/ S for active site
– Overcome w/ incr’d [S]
– Affects KM, not Vmax
![Page 27: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/27.jpg)
![Page 28: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/28.jpg)
Reversible Inhib’n (cont’d)• Uncompetitive
– Binds only when S already bound (so ES complex)
– Bind at site away from active site
– Causes conform’l change, E inactivated
– Not overcome w/ incr’d [S]
– Affects both KM, Vmax
– Common when S1 + S2
![Page 29: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/29.jpg)
![Page 30: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/30.jpg)
Reversible Inhib’n (cont’d)• Noncompetitive (Mixed)
– When S bound or not
– Bind at site away from active site
– Conform’l change in E
– E inact’d when I bound
– Decr’d E avail for binding S, rxn catalysis
– Not overcome w/ incr’d [S]
– Affects both KM, Vmax
– Common when S1 + S2
![Page 31: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/31.jpg)
![Page 32: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/32.jpg)
Effect of pH on Catalysis• Optimum pH where max activity
• Aa’s impt to catalysis must maintain partic ionization
• Aa’s in other parts of enz impt to maintain folding, structure must also maintain partic ionization
• Can predict impt aa’s by activity changes at different pH’s (use pKa info)
![Page 33: Enzyme Kinetics Chapter 6. Kinetics Study of rxn rates, changes with changes in experimental conditions Simplest rxn: S P –Rate meas’d by V = velocity.](https://reader036.fdocument.pub/reader036/viewer/2022081504/5697c02b1a28abf838cd84d8/html5/thumbnails/33.jpg)