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MoMF > Enzyme Kinetics and Inhibition > Flashcards

Enzyme Kinetics and Inhibition Flashcards

1
Q

Irreversible Reactions

A –> P

A

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]

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2
Q

Characteristics of 1st order reactions

A

Exponent is 1

Units: s^-1

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3
Q

Bimolecular irreversible reaction

A + B –> P

A

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]

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4
Q

Unimolecular reversible reaction

A P

A

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

^rate of P formation and rate of A disappearance

Rate gained = rate loss AT EQUILIBRIUM

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5
Q

Equilibrium constant: Keq

A

K1/k2 = [P]/ [A]

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6
Q

Steady state Assumption for Michaelis Menten

A

[ES] assumed to be unchanging

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

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7
Q

Formula for [ES] under steady state

A

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

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8
Q

Maximal Velocity

A

When E is saturate with S

[ES] = [E]t

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

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9
Q

Michaelis Menten equation

A

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

~ hyperbolic curve ~

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10
Q

of active sites are filled

A

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

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11
Q

Michaelis Menten Assumptions

A
  • 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]&raquo_space; [E]
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12
Q

Michaelis constant

A

Km= (k2 + k3)/ k1

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13
Q

Larger Km

A

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

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14
Q

Vm=k3[E]t

A

Maximum rate when [ES] = [E]t

Proportional to k3

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15
Q

Turnover number= k3=kcat

A
  • 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
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16
Q

Catalytic Efficiency- what happens when [S]

A

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

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17
Q

What is catalytic efficiency?

A

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

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18
Q

Kcat/Km

A

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

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19
Q

Perfect enzymes

A

Have the highest kcat/Km values

Limited only by the rate of diffusion of substrate to enzyme

10^8 - 10^9

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20
Q

Slowest step of the enzyme reaction

A

Diffusion of substrate to enzyme

However overall reaction is fast

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21
Q

Ideal substrate range

A

1/3 [KM] 2 [KM]

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22
Q

Line-weaver Burke

A

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

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23
Q

Dis-advantages to the Lineweaver Burk Plot

A

Distorts errors at low [S]

Compresses data at high [S]

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24
Q

Sequential Mechanism

A

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

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25
Q

Order sequential

A

Specific order for substrate binding and product leaving

“A has to go in first”

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26
Q

Random sequential

A

Random order for substrate binding and product leaving

“Either A or B can go into the reaction first”

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27
Q

Ping-Pong Mechanism (double replacement)

A

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

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28
Q

Reversible inhibition

A

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

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29
Q

Irreversible Inhibitors

A

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

Permanent inactivation of the enzyme

30
Q

Competitive Inhibition

A

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
Q

Analysis of Competitive Inhibitors

A

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
Q

Substrate analogs

A

Have key structural features that mimic the substrate

33
Q

Transition state analogs

A

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

34
Q

Uncompetitive Inhibitor

A

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
Q

How does a uncompetitive inhibitor work?

A

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
Q

Do the rate constants change during inhibition for uncompetitive inhibitors?

A

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

37
Q

How does the degree of inhibition change with uncompetitive inhibition?

A

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

38
Q

Noncompetitive Inhibition

A

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

Lacks structural resemblance to substrate

39
Q

How does noncompetitive inhibition work?

A

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

40
Q

Kinetics of Noncompetitive Inhibitors

A

Vm –> decrease

Km –> no change

Catalytic efficiency –> decrease

increasing [S] cannot overcome effects

41
Q

Degree of inhibition for noncompetitive inhibition?

A

Degrees of inhibition is constant with increasing [S]

42
Q

% Inhibition

A

(1-voi/vo) X 100

43
Q

Relative Rate

A

Voi/Vo

44
Q

Relative rate- Competitive Inhibition

[S]

A

1/(1+ [I]/Ki)

45
Q

Relative rate- Competitive Inhibition

[S]&raquo_space; Km

A

1

46
Q

Relative rate- Uncompetitive Inhibition

[S]

A

1

47
Q

Relative rate- Uncompetitive Inhibition

[S]&raquo_space; Km

A

1/(1+[I]/Ki)

48
Q

Relative Rate- Noncompetitive Inhibition

[S]

A

1/(1+[I]/Ki)

49
Q

Relative Rate- Noncompetitive Inhibition

[S]&raquo_space; Km

A

1/(1+[I]/Ki)

50
Q

Irreversible Inhibitors

A

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

51
Q

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

A

[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
Q

Irreversible Inhibitors- Group Specific Reagents

A

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

53
Q

Irreversible Inhibitors- Affinity Labels

A

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

More specific than group specific reagents

54
Q

Irreversible Inhibitors: Suicide Inhibitors

A

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
Q

Doe Allosteric Control obey Michaelis Menten model?

A

Does not obey Michaelis-Menten Kinetics

Shows a sigmoidal dependence of reaction velocity on substrate concentration

56
Q

When would you use allosteric control?

A

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
Q

How does Allosteric regulation work?

A

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
Q

Homotropic effector

A

Effector is same as substrate

Site is usually adjacent to active site (adjoining subunit)

Interaction almost always increases activity (sigmoidal curve)

59
Q

Heterotrophic Effector

A

Effector is different from substrate

Site is “true” allosteric site

Interaction increases or decreases activity

60
Q

How is allosteric control different from inhibition?

A

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

61
Q

What can allosteric control change?

A

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

62
Q

Positive Effector (A)

A

Binds to activator site and increases activity –> decreases Km

63
Q

Negative effector

A

Binds to inhibitory site and decreases activity –> increases Km

64
Q

How can allosteric enzymes by controlled?

A

Thru feedback inhibition

65
Q

Regulatory Proteins

A

Proteins that either have Stimulatory or inhibitory reversible interactions with enzymes

66
Q

Calmodulin

A

Regulatory protein that stimulates activity

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

67
Q

Antihemophilic factor (factor VIII)

A

Regulatory protein that stimulates activity

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

68
Q

PKA

A

Regulatory protein that inhibits activity

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

69
Q

How does reversible covalent modifications work?

A

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
Q

Proteolytic Activation

A

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

MoMF (25 decks)

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