Enzyme Kinetics 2

Question 1

Define “First order reaction”

Answer 1

A disappears as B is produced (A–>B)

Question 2

What are the 2 equations for velocity (v)?

Answer 2

v= - d[A]/ dt
and
v= d[B]/dt

Question 3

For a “first order reaction”, the rate is DIRECTLY proportional to…..?

Answer 3

[A] (rate is dependant on A)

Question 4

During the reversible reaction, AB, does the rate constant (k) change?

Answer 4

No

Question 5

During the reversible reaction, AB, does the velocity increase or decrease, and why?

Answer 5

Increase

• A is used up
• B become the substrate for the reverse reaction

Question 6

What are the conditions for a reaction to be in “equilibrium”? (2 answers)

Answer 6

• rates of forward and backwards reactions are EQUAL

- overall rate of reaction is zero

Question 7

Define “second order reaction”

Answer 7

based on conc. of 2 reactants

Question 8

What is assumed when considering second order reactions?

Answer 8

• rate forward reaction is linearly proportional to [A] and [B]
• the reverse reaction is linearly proportional to [C]

Question 9

What is the rate equation for a second order reaction?

Answer 9

d[A}/dt= rate of [C] - rate of [A][B]

Question 10

What is “equilibrium”?

Answer 10

the net rate of reaction is zero

Question 11

What is the formula for the equilibrium constant?

Answer 11

K{1}[C]= [A][B], K{1}= k{-1}/k{1}

Question 12

What is the “equilibrium constant” described as?

Answer 12

the extent of the reaction, NOT its speed

Question 13

In an enzymatic reaction, what limits the reaction out of the substrate and enzyme?

Answer 13

the enzyme

Question 14

What is the general process for enzyme catalysis?

Answer 14

• the enzyme binds to the substrate to form the intermediate (ES) in a REVERSIBLE reaction
• the intermediate ES decomposes IRREVERSIBLY to produce the product and leave the enzyme (not changed)

Question 15

What is the enzyme catalysis mechanism made up of?

Answer 15

two consecutive reactions with reversible 1st step

Question 16

What is assumed during enzyme catalysis? (3 answers)

Answer 16

• [S] > [E] at the beginning (initial)
• [ES] remains constant
• [E] is limiting (constant inc. of P)

Question 17

The initial rate increases with…?

Answer 17

[E] and [S]- leaves off (satuarates) to Vmax ([S])

Question 18

What is “Km”?

Answer 18

• [S] at 1/2 Vmax

- rate constant describing the affinity of the enzyme for the substrate (dissociation of the ES complex)

Question 19

What is the proposed mechanism for the saturating reaction rate?

Answer 19

k1 k2
E+S ES–>E+P
k-1

Question 20

What assumptions are made when considering the saturating reaction rate? (2 answers)

Answer 20

• the rate of ES FORMATION= the rate of ES dissociation

- ES reaches equilibrium quickly (k1/k-1 ignored)

Question 21

What is the rate equation considering the constant Km?

Answer 21

v= k2[Eo][S] / [S] + Km

Question 22

The higher the Km value…

Answer 22

…the lower the affinity

Question 23

Define “Vmax”

Answer 23

the maximum catalytic rate at full saturation

Question 24

What is assumed about the conc. of P?

Answer 24

it’s practically zero

Question 25

The reaction velocity (Vo) varies with...

Answer 25

... [S]

Question 26

The curve approaches Vmax...

Answer 26

... asymptotically

Question 27

What is a Lineweaver Burke plot?

Answer 27

plotting 1/v against 1/[S] (double reciprocal)

Question 28

Why is a Lineweaver Burke plot useful?

Answer 28

more accurate as it generates a straight line

Question 29

How is the Vmax estimated using the Lineweaver-Burke plot?

Answer 29

the y-intercept when x is 0

Question 30

How is the Km estimated using the Lineweaver-Burke plot?

Answer 30

the -x axis when y is 0

Question 31

What is a disadvantage of Lineweaver-Burke plots?

Answer 31

the plot is dominated by points at low [S]- less accurate

Question 32

What are the 2 types of enzyme inhibitors?

Answer 32

- competitive | - non-competitive

Question 33

Describe "competitive inhibition"

Answer 33

- inhibitor binds to the active site of the enzyme | - reliant on [S] and [inhibitor]

Question 34

Describe "non-competitive inhibition"

Answer 34

- inhibitor binds elsewhere on the enzyme | - NOT reliant on conc.

Question 35

What is "Ki"?

Answer 35

the dissociation constant for an inhibitor

Question 36

What are the kinetics of competitive inhibitors?

Answer 36

- Vmax remains the same (does not change) - Km is increased (lower affinity for the enzyme) - increasing [S] overcomes the inhibition; reliant on conc.

Question 37

Why is the Vmax unaltered and the Km increased? | double reciprocal graph

Answer 37

- the Vmax cross at the same point on the y-axis when x=0 | - the Km values cross at 2 different points on the x-axis when y=0

Question 38

What are the kinetics of non-competitive inhibitors?

Answer 38

- Vmax lowers - Km is the same - less E available - increasing [S] DOES NOT overcome the inhibition; NOT reliant on conc.

Question 39

Why is the Vmax decreased and the Km unaltered?

Answer 39

- the Vmax crosses at different points on the y-axis when x=0 - the Km values cross at the same point on the -x-axis when y=0

Question 40

What other route can the reaction take when a competitive inhibitor is present?

Answer 40

E+IEI

Question 41

What other route can the reaction take when a NON-competitive inhibitor is present?

Answer 41

E+IEI+S-->EIS

Question 42

What are the Km and Vmax values of non-competitive inhibitors?

Answer 42

- Km= unaltered | - Vmax= decreased

Question 43

What does the "non-competitive inhibitor" and "no inhibitor present" graph show? (double reciprocal graph)

Answer 43

x-axis (1/[S]): the 2 lines have the same Km value (on negative scale when y=0) y-axis (1/V): the 2 lines have 2 different Vmax values (on positive scale when x=0)

Question 44

What occurs when a competitive inhibitor binds to an enzyme?

Answer 44

- [I] binds to free [E] only - it competes with [S] - inc. [S] overcomes inhibition by [I]

Question 45

What occurs when a non-competitive inhibitor binds to an enzyme?

Answer 45

- [I] binds to free [E] or [ES] complex | - inc. [S] can not overcome [I] inhibition

Question 46

What are the other plots?

Answer 46

- Eadie Hofstee | - Hanes and Dixon

Question 47

What is the Eadie Hofstee plot?

Answer 47

- plot v against v/[S]-- linear - gradient: Km - y-intercept: Vmax

Question 48

What is the Hanes or Dixon plot?

Answer 48

- plot [S]/v against [S]-- linear - gradient: 1/Vmax - y-intercept: Km/Vmax

Question 49

What do errors in v lead to in the Eadie Hofstee or Hanes plots?

Answer 49

non-linearity

Question 50

What are competitive inhibitors?

Answer 50

substrate "mimic"

Question 51

What is the active site of an enzyme?

Answer 51

the region that binds the substrates (and cofactors)

Question 52

What are the catalytic groups of an active site?

Answer 52

residues that directly participate in making and breaking bonds

Question 53

What does the interaction of the enzyme and substrate active site promote?

Answer 53

the formation of the transition state

Question 54

What most directly lowers the delta G of the reaction?

Answer 54

the active site | - resulting in rate enhancement of the reaction

Question 55

What are the 5 common features of active sites?

Answer 55

1) active sites= 3D cleft formed by groups-- from different parts of amino acid sequence 2) the active site takes up a relatively small part of the total volume of an enzyme 3) active sites are clefts or crevices 4) substrates bound to enzymes by multiple weak attractions 5) specificity of binding depends on precisely defined arrangement of atoms at active site

Question 56

What is the specificity of an enzyme?

Answer 56

- the precise interaction of substrate with enzyme | - result of intricate 3D structure of enzyme protein

Question 57

What is enzyme inhibition by DIPF?

Answer 57

DIPF reacts with Ser in acetylcholinesterase which inactives the enzyme

Question 58

What does DIPF stand for?

Answer 58

Diisopropylphosphofluoridate