Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf ·...
Transcript of Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf ·...
![Page 1: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/1.jpg)
Enzyme Kinetics 2018DR BELA GOYAL
![Page 2: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/2.jpg)
Learning Objectives
Enzyme Kinetics
Enzyme Inhibition
Drugs utilizing kinetics and inhibition and its clinical utility
![Page 3: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/3.jpg)
Enzyme Kinetics
the quantitative measurement of the rates of enzyme-catalyzed reactions
and the systematic study of factors that affect these rates
![Page 4: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/4.jpg)
Catalysts
Increase rate of reaction by factor of 106
Highly selective and specific
Not changed as a result of catalysis
Does not change the equilibrium constant
Enzymes Alter Only the Reaction Rate
and Not the Reaction Equilibrium
![Page 5: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/5.jpg)
Factors affecting reaction velocity
Substrate concentration
The rate of an enzyme-catalyzed reaction increases with substrate
concentration until a maximal velocity (Vmax) is reached
![Page 6: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/6.jpg)
Temperature
Bell shaped curve
Increase of velocity with temperature
Decrease of velocity with higher temperature
![Page 7: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/7.jpg)
pH
Bell shaped curve
Effect of pH on the ionization of the active site
Effect of pH on enzyme denaturation
Variable pH optimum
![Page 8: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/8.jpg)
Kinetic Order of Reaction
Sum of the molar ratios of the reactants defines the kinetic order of the
reaction
First order
Second order
Pseudofirst order reaction
![Page 9: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/9.jpg)
Michaelis Menten Equation
Relationship between initial velocity and substrate concentration
[Vo]
[S]
Vmax
Km
1/2Vmax
![Page 10: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/10.jpg)
Km and its importance
The Michaelis constant Km is the substrate concentration
at which Vi is half the maximal velocity (Vmax/2) attainable
at a particular concentration of the enzyme.
Unit?
Reflects the affinity of the enzyme for that substrate:
inverse relationship
Specific for enzyme substrate combination
Order of reaction
![Page 11: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/11.jpg)
Clinical importance of Km
Hexokinase vs Glucokinase
![Page 12: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/12.jpg)
Line-Weaver Burk plot
Double Reciprocal Graph
Linear curve
1 = Km + 1
V0 Vmax [S] Vmax
Linear form of Michaelis menten equation to
Determine Km and Vmax.
![Page 13: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/13.jpg)
Units of Enzyme activity
Amount of substrate converted to product per unit time under standard
conditions of pH and Temperature
IUB unit: Katal (µmol/min)
SI Unit: (mol/sec)
![Page 14: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/14.jpg)
Relative activities of Enzymes
Specific activity (Vmax/protein concentration): Impure Enzymes
Turnover number(Vmax/moles of enzyme): Homogenous Enzymes
Catalytic constant, Kcat [Vmax/No. of active sites(St)]: unit time-1
Catalytic efficiency: Kcat/Km (Carbonic anhydrase, ADA,
acetylylcholinesterase)
![Page 15: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/15.jpg)
Two substrate Reactions(Bi BiReactions)
Sequential : Random
Ordered
Ping Pong: Double displacement reactions
![Page 16: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/16.jpg)
Two substrate Reactions
Random
Ordered/Sequential
Ping Pong/Double Displacement
![Page 17: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/17.jpg)
Enzyme Inhibition
![Page 18: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/18.jpg)
Types of Inhibitions based on kinetics
Competitive Inhibition
Non-Competitive Inhibition
Un-Competitive inhibition
![Page 19: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/19.jpg)
Competitive Inhibition
Binding at substrate binding site
Inhibitor similar to substrate
Km increased
Vmax same
![Page 20: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/20.jpg)
Clinical Application/Drugs
Statin Drugs
Competitive Inhibitors of HMG CoA reductase
Sulpha Drug (Str. Analogues of PABA)
Inhibits Folic acid synthesis in Bacteria
Methanol Poisoning
![Page 21: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/21.jpg)
Non-Competitive Inhibition
Substrate and inhibitor binds at different sites
Not structural analogues
Decrease Vmax
Km same
![Page 22: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/22.jpg)
Drugs/Toxins based on Non -
Competitive Inhibition
Ferrochelatase (Inhibition by Lead)
Acetylcholinesterase (Insecticides)
Cytochrome oxidase(Cyanide)
![Page 23: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/23.jpg)
Uncompetitive Inhibition
Inhibitor binds to ES Complex
Both Km and Vmax decreases 1/V
1/S
With Inhibitor
![Page 24: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/24.jpg)
Examples of Drugs showing
Uncompetitive Inhibition
Lithium (Inositol monophosphatase)
Phenylalanine(Placental ALP)
![Page 25: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/25.jpg)
Classification based on Reversibility
Reversible
Irreversible: Chemical modification or Covalent modification
![Page 26: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/26.jpg)
Irreversible inhibitors Poison Enzymes
Diisopropylflurophosphate (nerve gas): covalently binds
acetylcholinestrase
Aspirin(Cox)
Penicillin (bacterial transpeptidase)
![Page 27: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/27.jpg)
Mechanism Based Inhibition
Suicide Inhibition
Contains chemical group that is transformed by catalytic machinery
Generates highly reactive group
Binds covalently to catalytically essential residues
![Page 28: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/28.jpg)
Drugs based on Suicide Inhibition
Allopurinol (inhibits xanthine oxidase: Oxypurinol)
5 fluorouracil (inhibits thymidylate synthase: FdUMP)
![Page 29: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/29.jpg)
Transition state Analogs & Abzymes
Transition state analog: A molecule with shape
similar to transition state
Catalytic Antibodies
Abzymes created using transition state analog
as antigens
![Page 30: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/30.jpg)
Clinical Scenario 1
A 45-year-old man presents to emergency with bradycardia, blurred vision,
vomiting, increased and salivation. He is a farmer using OPC Spray for his field
and pipe ruptured. Type of inhibition?
(A) Competitive
(B) Noncompetitive
(C) Uncompetitive
(D) Irreversible
![Page 31: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/31.jpg)
Clinical scenario 2
A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,
dysphagia and slurred speech. A drug is administered with following effect.
What is true
a. Competitive: Vmax same, km increased
b. Competitive : Vmax same, km decreased
c. Non-competitive: Vmax decreased, Km same
d. Non-competitive: Vmax decreased, Km decreased
1/V
1/S
In presence of drug
![Page 32: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/32.jpg)
Clinical Scenario 3
A patient wants to go to Manali for trekking. He took a medicine for mountain
sickness with following kinetics. What is the type of inhibition?
A. Competitive
B. Noncompetitive
C. Uncompetitive
D. Allosteric
1/V
1/S
With Drug
![Page 33: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/33.jpg)
![Page 34: Enzyme Kinetics 2018 - AIIMS RISHIKESHaiimsrishikesh.edu.in/.../10/831_Enzyme_Kinetics.pdf · Clinical scenario 2 A 35 year old lady comes to OPD with evening fatigue, eyelid drooping,](https://reader036.fdocuments.us/reader036/viewer/2022070808/5f0694c97e708231d418b342/html5/thumbnails/34.jpg)
References
Victor W. Rodwell, David A. Bender, Kathleen M.
Botham, Peter J. Kennelly, P. Anthony Weil. Harper’s
Illustrated Biochemistry, 30th Edition
Denise R. Ferrier; Lippincott Illustrated Reviews
Biochemistry, 7th Edition
Thank You!