Biochem Enzymes Lecture 2 Flashcards
We study enzyme catalyzed reactions though _______
Enzyme Kinetics
In enzyme kinetics, we want to determine how ______ changes in response to experimental variables
Reaction rate
Substrate Concentration is important to rate determination, but [S] changes during the reaction as it is converted to product. We can simplify the problem by measuring the _____
Initial rate (initial velocity Vo)
What is the relationship of [S] and [E] at the start of a reaction?
So the [S] is thought of as
[S]»[E]
Constant
Vo (initial rate) can be investigated as a function of ___, which is varied by the experimenter
[S]
At low [S], Vo increases/decreases in an ~approximately linear fashion with an increase in [S].
Increases
At a higher [S], Vo increases become SMALLER/LARGER
Smaller until a plateau is reached
This is close to Vmax on the substate concentration and initial velocity curve
Plateau region
From the kinetic behavior in the graph figure, a theory was proposed
In a fast first step, the enzyme combines with its substrate to form….
In slower second step, the ES complex does what?
An enzyme substrate complex
Breaks down to give free enzyme and reaction product P
The slower reaction step that limits the overall reaction rate is called the….
Therefore, the overall rate must be proportional to the concentration of species that reacts in which step?
Rate Limiting Step
The slower second step
At low [S], the reaction rate is proportional to
[S] because the equilibrium favors formation
of ES as [S] increases
!
Vmax is reached when virtually all of the
enzyme is present as the…..
ES complex (no free enzyme)
When all of the enzyme is present as the ES complex, we can say that the enzyme is _____ with substrate. It is responsible for the plateau in the Vo vs. [S] curve
Saturated
The graph is an example of ___ kinetics. After the ES complex breaks down to P, the free enzyme can catalyze another reactant molecule
Note the a ____ state, microseconds long, occurs where ES initially builds up. The reaction quickly achieves a ___ state, where the [ES] is approximately constant
Saturation Kinetics
Pre-steady state
Steady State
E+S —> ES—-> E+P
Important equations
The measured Vo reflects the ______ state.
Analysis of initial rates are _______
Steady State
Steady state kinetics
The curve in fig. 6-11 can be expressed algebraically by which equation?
Michaelis-Menten Equation
What is the Michaelis Menten equation?
Vo = Vmax[S]/(Km + [S])
All of the terms in the Michaelis-Menten equation can be measured experimentally
!
Early in the reaction, the concentration of what is negligible?
P, so P—->S can be ignored
Vo is determined by the breakdown of ___ to ____
Therefore, Vo=?
[ES] to [P]
Vo = k2[ES]
Since the concentration of ES is not measured easily, the Vo = k2[ES] equation must be expressed differently
We begin by finding what?
The concentration of the enzyme
[total enzyme]
How is the total enzyme concentration expressed?
Therefore, how do you solve for free Enzyme?
Also, [S]» [Et], so the amount of substrate
bound by ES is negligible compared to [S]
[Et] = [E] + [ES]
[E] = [Et] –[ES]
!
In step 1, what is the rate of ES formation?
What is the rate of ES breakdown?
=k1([Et] – [ES])[S]
=k-1[ES] + k2[ES]
In step two, what is the equation representing the steady state assumption. It represents that the rate of ES formation = the rate of ES breakdown
k1([Et] – [ES])[S] = k-1[ES] + k2[ES]
What is the final equation that allows you to solve for [ES]
[ES] = k1[Et][S]/(k1[S] + k-1 + k2)
What is the Michaelis-menton equation?
There were other steps to get to this equation. Do you need to know?
Vo = Vmax [S]/(Km + [S])
The Michaelis-Menten equation is the rate equation for an enzyme catalyzed reaction that has how many substrates?
One.
When Vo=1/2Vmax, Vmax/2 =?
Therefore, we can divide by Vmax and get what equation? Important equation!
Vmax[S]/(Km + [S])
Km=[S] when Vo=1/2Vmax
All enzymes that exhibit a ____ dependence of Vo on [S] follow Michaelis-Menten Kinetics
Hyperbolic dependence
This is the assumption that after a very short time, the concentration of ES remains constant because ES formation = ES brekdown
Steady State Assumption
Is the steady state assumption in the Michaelis-Menten equation?
Yes. Analysis of Vo are steady state kinetics
When does Vmax occur?
When all of the E is in the form of an ES complex
The Michaelis-Menten equation does NOT depend on
the two- step reaction mechanism proposed by
Michaelis and Menten as follows:
FIND THE EQUATION IN THE BOOK!
What is the important limitation of the steady state approach
the meaning of Vmax and Km can be different for different enzymes
A more convenient plot than the one in figure 6-12 for approximating Km is a double reciprocal plot, known as a ___ plot
Lineweaver-Burk plot
What are the axes of the Lineweaver-Burk plot?
For enzymes which obey Michaelis-Menten kinetics, what shape is the data?
What is the slope equal to?
What is the Y intercept?
What is the X intercept?
1/Vo and 1/[S]
A straight line
Km/Vmax
1/Vmax
-1/Km
This linear plot gives a more accurate measure of Vmax, which is only approximated in the simple plot of Vo vs. [S]
The Km values for some substrates and enzymes are shown in table 6-6
The data in the table shows that Km is different for different enzymes and even varies for different substrates of the same enzyme
For example, D-glucose and D-fructose are both
substrates for the enzyme hexokinase, but there Km
values are different (0.05 and 1.5 mM, respectively).
A kinetic analysis gives important information about the ___ of an enzyme catalyzed reaction
For example, the substrate must form a complex with the enzyme before it can be converted to products. Complex formation is RESERVISBLE/IRREVERSIBLE
Mechanism
reversible
The actual meaning of Km depends upon the number and relatives rates of the individual steps of the reaction mechanism
What is the equation for Km in reactions with 2 steps?
Km=(k2 + k-1)/k1
If K2
k-1/k1
KD
Affinity
Smaller
In some cases, Michaelis-Menten kinetics still apply but Km is not a measure of affinity
Sometimes k2»k-1 and Km=….
Sometimes Km remains a complex function of all 3 k values
Often, the reaction goes through multiple steps after ES formation and many rate constants define Km
Km= k2/k1
In another overall reaction for Km, if the product release is rate limiting, then most of the enzyme is present in the ___ form at saturation, and Vmax = ?
____, a more general rate consant, can be used to define the limiting rate of an enzyme catalyzed reaction at saturation
EP form at saturation
Vmax = k3[Et]
Kcat
In the M-M equation, what is the formula for Kcat?
Therefore, what is the equation for Vo?
Vmax/[Et]
Vo=kcat[Et][S]/(Km + [S])
Kcat is a ___ order rate constant and is called the ____ number
first order
Turnover number
This value is defined as the number of substrate molecules converted to product in a given time on one enzyme molecule when the enzyme is saturated with substrate
Kcat
The best way to compare the turnover of different
substrates by the same enzyme or the catalytic
efficiencies of different enzymes is to compare the ratio of ____ to _____
This ratio is the
ratio of kcat/km
Specificity Constant
What is the rate constant for conversion of E+ S to E+ P?
Kcat/Km
When [S]«Km, then Vo =?
What order is this rate equation?
What are the units?
There is a diffusion controlled limit for this rate constant, of ___ to ____
Vo = (kcat/ Km) [Et][S]
Second order
M-1s-1
10^8 to 10^9
T/F Many enzymes catalyze reactions with
multiple substrates
True
In the reaction catalyzed by hexokinase:
ATP + glucose → ADP + glucose 6-phosphate
______ kinetics can be used to analyze the rates of this bisubstrate reaction
Enzymes with two substrates usually involve a transfer of an atom or ______ from one
substrate to a different one.
Michaelis Menten kinetics
Functional group
What are the different pathways of enzymes with two substrates may proceed by?
Ternary-complex
Ping-Pong (double displacement)
