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Flashcards in Enzyme Kinetics and Inhibition Deck (70):
1

Irreversible Reactions

A --> P

Rate of P formation equals rate of A disappearance

Rate of P formation is directly proportional to the concentration of reactant

V= dp/dt= k[A]

V= -dA/dt= k[A]

2

Characteristics of 1st order reactions

Exponent is 1

Units: s^-1

3

Bimolecular irreversible reaction

A + B --> P

Rate of P formation equals rate of disappearance of A OR B

Rate of P formation (or A/B disappearance) is directly proportional to concentration of reactants

V= dp/dt = k [A] [B]

4

Unimolecular reversible reaction

A P

V= dp/dt = k1[A] - k2[P]

^rate of P formation and rate of A disappearance

Rate gained = rate loss AT EQUILIBRIUM

5

Equilibrium constant: Keq

K1/k2 = [P]/ [A]

6

Steady state Assumption for Michaelis Menten

[ES] assumed to be unchanging

Michaelis Constant=
Km= (k2 +k3)/k1 > [E] and ES formation has negligible effect on S... [S]= constant = [S]t

7

Formula for [ES] under steady state

[ES]= [E][S]/ Km = ([E]t [S])/ (Km + [S])

8

Maximal Velocity

When E is saturate with S

[ES] = [E]t

Vo= k3 [ES] = k3 [E]t = Vm

9

Michaelis Menten equation

V = Vm [S]/ (Km + [S])

~ hyperbolic curve ~

10

# of active sites are filled

[ES]/ [E]t = v/Vm = [S]/ (Km + [S])

11

Michaelis Menten Assumptions

-Formation of ES complex between enzyme and substrate

-no back reaction from product buildup (k4=0)

-initial velocities used for analysis (t=0)

-steady state for [ES]

-negligible depletion of substrate [S] >> [E]

12

Michaelis constant

Km= (k2 + k3)/ k1

13

Larger Km

Has a smaller v at the same [S]

Graph levels off at the same Vm but reaches it slower

Weak binding of the [ES] --> low affinity

14

Vm=k3[E]t

Maximum rate when [ES] = [E]t
Proportional to k3

15

Turnover number= k3=kcat

-Catalytic ability

-Typical values: 1-10^4 s^-1

-Number of S molecules converted to P by one E molecule in unit time under saturation conditions

-larger kcat --> larger v --> faster reaction

16

Catalytic Efficiency- what happens when [S]

Typical physiological conditions

Plot of v versus [S] is learn with an apparent second order rate constant: k3/Km = kcat/Km --> proportional to that initial slope Vm/Km

17

What is catalytic efficiency?

Kcat/Km --> how well an enzyme reacts with dilute amounts of substrate

18

Kcat/Km

Combines attributes of kcat and Km (characteristics of E-S interaction)

19

Perfect enzymes

Have the highest kcat/Km values

Limited only by the rate of diffusion of substrate to enzyme

10^8 - 10^9

20

Slowest step of the enzyme reaction

Diffusion of substrate to enzyme

However overall reaction is fast

21

Ideal substrate range

1/3 [KM] 2 [KM]

22

Line-weaver Burke

Take double reciprocal of the MM equation

1/v = Km/Vm (1/[S] + 1/Vm

Slope= Km/Vm = Km/(kcat [E]t)

Y-intercept= 1/Vm

X-intercept= -1/Km

Low slope has a better catalytic efficiency

23

Dis-advantages to the Lineweaver Burk Plot

Distorts errors at low [S]

Compresses data at high [S]

24

Sequential Mechanism

Substrate bind to form a ternary complex with the enzyme before product is release

25

Order sequential

Specific order for substrate binding and product leaving

"A has to go in first"

26

Random sequential

Random order for substrate binding and product leaving

"Either A or B can go into the reaction first"

27

Ping-Pong Mechanism (double replacement)

One substrate binds and release product before second substrate binds and release product

A goes in P comes out * enzyme intermediate* B goes in and Q comes out

28

Reversible inhibition

Bind the enzyme with noncovalent interactions
Dissociate rapidly
Allow the enzyme to recover its original activity

29

Irreversible Inhibitors

Bind the enzyme with covalent interactions targeting a critical residue for catalysis

Permanent inactivation of the enzyme

30

Competitive Inhibition

Binds to only the free enzyme
Compete with substrate for active site

Usually resembles shape and structure of substrate (or transition state) --> lacks functionality for reaction

Hinders reaction by interfering with substrate binding and reducing amount of ES complex

31

Analysis of Competitive Inhibitors

reduced rate is same as noncompetitive inhibitor at very low [S]

Degree of inhibition decreases with increasing [S] --> low substrate concentration is the best place for the inhibitor to work

Km decreases

Vm stays the same

Kcat/Km decreases

32

Substrate analogs

Have key structural features that mimic the substrate

33

Transition state analogs

Stable compounds that resemble the transition state in structure and polarity or charge

34

Uncompetitive Inhibitor

Binds only to ES Complex (substrate must be bound to enzyme) at a site different from active site but created by substrate-enzyme interaction

Lacks structural resemblance to substrate

35

How does a uncompetitive inhibitor work?

Hinders reaction by distorting active site and making catalytically inactive

^^catalytic residues cannot line up properly

Km decreases by the same factor of Vm

Vm decreases by the same factor of Km

Catalytically efficiency stays the same (same slope)

36

Do the rate constants change during inhibition for uncompetitive inhibitors?

Rate constants don't change, we're looking at them when the inhibitors are present --> called apparent changes

37

How does the degree of inhibition change with uncompetitive inhibition?

Doesn't do much as low [S] (rate is same as control) but inhibition increases with increasing [S]

38

Noncompetitive Inhibition

Binds with same Ki to free enzyme or ES complex at site different from active site

Lacks structural resemblance to substrate

39

How does noncompetitive inhibition work?

Hinders reaction by distorting enzyme structure and preventing alignment of catalytic center

40

Kinetics of Noncompetitive Inhibitors

Vm --> decrease

Km --> no change

Catalytic efficiency --> decrease

*increasing [S] cannot overcome effects*

41

Degree of inhibition for noncompetitive inhibition?

Degrees of inhibition is constant with increasing [S]

42

% Inhibition

(1-voi/vo) X 100

43

Relative Rate

Voi/Vo

44

Relative rate- Competitive Inhibition

[S]

1/(1+ [I]/Ki)

45

Relative rate- Competitive Inhibition

[S] >> Km

1

46

Relative rate- Uncompetitive Inhibition

[S]

1

47

Relative rate- Uncompetitive Inhibition

[S] >> Km

1/(1+[I]/Ki)

48

Relative Rate- Noncompetitive Inhibition

[S]

1/(1+[I]/Ki)

49

Relative Rate- Noncompetitive Inhibition

[S] >> Km

1/(1+[I]/Ki)

50

Irreversible Inhibitors

Result in permanent inactivation by forming stable covalent bonds with functional groups involved in enzyme activity

51

What happens to [E]t and Vm with irreversible inhibitors?

[E]t decreases Vm decreases but no change in Km
^this is because inhibitors remove free enzymes from the reaction

Operate within the active site

52

Irreversible Inhibitors- Group Specific Reagents

Group specific reagents covalently interact with specific side chains of enzyme residues

53

Irreversible Inhibitors- Affinity Labels

Structurally similar to substrate and covalently bind with active-site residues

More specific than group specific reagents

54

Irreversible Inhibitors: Suicide Inhibitors

Also called mechanism-based inhibitors or suicide in activators

Bind at the active site and "trick" the enzyme into activating the catalytic mechanism

A chemically reactive intermediate is produced which covalently modifies the enzyme and results in permanent inactivation

^^permanent inactivation of the enzyme is a result of the enzyme's own participation

55

Doe Allosteric Control obey Michaelis Menten model?

Does not obey Michaelis-Menten Kinetics

Shows a sigmoidal dependence of reaction velocity on substrate concentration

56

When would you use allosteric control?

These enzymes are typically rate-determining enzymes in metabolic pathways or at a junction where the substrate can be used for more than one pathway

57

How does Allosteric regulation work?

Involves noncovalent binding of a ligand (effector) to a site other than the active site (Regulatory or allosteric site) --> this binding affects the activity of the active site

Allosteric enzymes are generally oligomers (>1 subunit) such that interaction on one subunit affects the others (+ or - cooperativity)

58

Homotropic effector

Effector is same as substrate

Site is usually adjacent to active site (adjoining subunit)

Interaction almost always increases activity (sigmoidal curve)

59

Heterotrophic Effector

Effector is different from substrate

Site is "true" allosteric site

Interaction increases or decreases activity

60

How is allosteric control different from inhibition?

Allosteric control actually changes the rate constants instead of the apparent change

61

What can allosteric control change?

Effector-induced conformational changes in the enzyme can alter activity by changing Km (k class) and Vm (v class) or both

62

Positive Effector (A)

Binds to activator site and increases activity --> decreases Km

63

Negative effector

Binds to inhibitory site and decreases activity --> increases Km

64

How can allosteric enzymes by controlled?

Thru feedback inhibition

65

Regulatory Proteins

Proteins that either have Stimulatory or inhibitory reversible interactions with enzymes

66

Calmodulin

Regulatory protein that stimulates activity

Senses intracellular Ca2+ concentration and activates proteins when calcium levels rise

67

Antihemophilic factor (factor VIII)

Regulatory protein that stimulates activity

Enhances activity of a serine protease to accelerate the blood clotting cascade

68

PKA

Regulatory protein that inhibits activity

Inhibitors PKA catalytic subunits until binding of cAMP causes dissociation of regulatory and catalytic subunits

69

How does reversible covalent modifications work?

Catalytic properties of enzymes are modified by adding/removing charged groups (phosphate, sulfate, acetate) --> that causes conformational change and alter their function

most common: the phosphorylation/dephosphorylation cycle of specific serine, threonine, and tyrosine residues

Rate depends on the concentration of kinases (phosphate example)

70

Proteolytic Activation

Many enzymes are synthesized as an inactive precursor called a pro enzyme or zymogen