Enzyme Kinetics 2 Flashcards
Define “First order reaction”
A disappears as B is produced (A–>B)
What are the 2 equations for velocity (v)?
v= - d[A]/ dt
and
v= d[B]/dt
For a “first order reaction”, the rate is DIRECTLY proportional to…..?
[A] (rate is dependant on A)
During the reversible reaction, AB, does the rate constant (k) change?
No
During the reversible reaction, AB, does the velocity increase or decrease, and why?
Increase
- A is used up
- B become the substrate for the reverse reaction
What are the conditions for a reaction to be in “equilibrium”? (2 answers)
- rates of forward and backwards reactions are EQUAL
- overall rate of reaction is zero
Define “second order reaction”
based on conc. of 2 reactants
What is assumed when considering second order reactions?
- rate forward reaction is linearly proportional to [A] and [B]
- the reverse reaction is linearly proportional to [C]
What is the rate equation for a second order reaction?
d[A}/dt= rate of [C] - rate of [A][B]
What is “equilibrium”?
the net rate of reaction is zero
What is the formula for the equilibrium constant?
K{1}[C]= [A][B], K{1}= k{-1}/k{1}
What is the “equilibrium constant” described as?
the extent of the reaction, NOT its speed
In an enzymatic reaction, what limits the reaction out of the substrate and enzyme?
the enzyme
What is the general process for enzyme catalysis?
- 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)
What is the enzyme catalysis mechanism made up of?
two consecutive reactions with reversible 1st step
What is assumed during enzyme catalysis? (3 answers)
- [S] > [E] at the beginning (initial)
- [ES] remains constant
- [E] is limiting (constant inc. of P)
The initial rate increases with…?
[E] and [S]- leaves off (satuarates) to Vmax ([S])
What is “Km”?
- [S] at 1/2 Vmax
- rate constant describing the affinity of the enzyme for the substrate (dissociation of the ES complex)
What is the proposed mechanism for the saturating reaction rate?
k1 k2
E+S ES–>E+P
k-1
What assumptions are made when considering the saturating reaction rate? (2 answers)
- the rate of ES FORMATION= the rate of ES dissociation
- ES reaches equilibrium quickly (k1/k-1 ignored)
What is the rate equation considering the constant Km?
v= k2[Eo][S] / [S] + Km
The higher the Km value…
…the lower the affinity
Define “Vmax”
the maximum catalytic rate at full saturation
What is assumed about the conc. of P?
it’s practically zero
The reaction velocity (Vo) varies with…
… [S]
The curve approaches Vmax…
… asymptotically
What is a Lineweaver Burke plot?
plotting 1/v against 1/[S] (double reciprocal)
Why is a Lineweaver Burke plot useful?
more accurate as it generates a straight line
How is the Vmax estimated using the Lineweaver-Burke plot?
the y-intercept when x is 0
How is the Km estimated using the Lineweaver-Burke plot?
the -x axis when y is 0
What is a disadvantage of Lineweaver-Burke plots?
the plot is dominated by points at low [S]- less accurate
What are the 2 types of enzyme inhibitors?
- competitive
- non-competitive
Describe “competitive inhibition”
- inhibitor binds to the active site of the enzyme
- reliant on [S] and [inhibitor]
Describe “non-competitive inhibition”
- inhibitor binds elsewhere on the enzyme
- NOT reliant on conc.
What is “Ki”?
the dissociation constant for an inhibitor
What are the kinetics of competitive inhibitors?
- Vmax remains the same (does not change)
- Km is increased (lower affinity for the enzyme)
- increasing [S] overcomes the inhibition; reliant on conc.
Why is the Vmax unaltered and the Km increased?
double reciprocal graph
- 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
What are the kinetics of non-competitive inhibitors?
- Vmax lowers
- Km is the same
- less E available
- increasing [S] DOES NOT overcome the inhibition; NOT reliant on conc.
Why is the Vmax decreased and the Km unaltered?
- 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
What other route can the reaction take when a competitive inhibitor is present?
E+IEI
What other route can the reaction take when a NON-competitive inhibitor is present?
E+IEI+S–>EIS
What are the Km and Vmax values of non-competitive inhibitors?
- Km= unaltered
- Vmax= decreased
What does the “non-competitive inhibitor” and “no inhibitor present” graph show?
(double reciprocal graph)
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)
What occurs when a competitive inhibitor binds to an enzyme?
- [I] binds to free [E] only
- it competes with [S]
- inc. [S] overcomes inhibition by [I]
What occurs when a non-competitive inhibitor binds to an enzyme?
- [I] binds to free [E] or [ES] complex
- inc. [S] can not overcome [I] inhibition
What are the other plots?
- Eadie Hofstee
- Hanes and Dixon
What is the Eadie Hofstee plot?
- plot v against v/[S]– linear
- gradient: Km
- y-intercept: Vmax
What is the Hanes or Dixon plot?
- plot [S]/v against [S]– linear
- gradient: 1/Vmax
- y-intercept: Km/Vmax
What do errors in v lead to in the Eadie Hofstee or Hanes plots?
non-linearity
What are competitive inhibitors?
substrate “mimic”
What is the active site of an enzyme?
the region that binds the substrates (and cofactors)
What are the catalytic groups of an active site?
residues that directly participate in making and breaking bonds
What does the interaction of the enzyme and substrate active site promote?
the formation of the transition state
What most directly lowers the delta G of the reaction?
the active site
- resulting in rate enhancement of the reaction
What are the 5 common features of active sites?
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
What is the specificity of an enzyme?
- the precise interaction of substrate with enzyme
- result of intricate 3D structure of enzyme protein
What is enzyme inhibition by DIPF?
DIPF reacts with Ser in acetylcholinesterase which inactives the enzyme
What does DIPF stand for?
Diisopropylphosphofluoridate
