Chapter 8: Kinetics Flashcards
What is kinetics the study of?
reaction rates
in a kinetic graph of time vs concentration, what does the slope tell you?
the rate (velocity) at which substrates are being consumed and/or rate (velocity) at which products are being formed
in a kinetic graph of time vs concentration, what does it mean when the graph plateaus/forms an asymptote?
equilibrium has been achieved
in a kinetic graph of VELOCITY vs concentration, what does it mean when the graph plateaus/forms an asymptote?
maximum velocity has been achieved
rxn can’t go any faster unless you add more enzyme
in a kinetic graph of VELOCITY vs concentration, what does the graph’s plateau/asymptote tell you about the reaction rate/velocity?
that it can’t go any faster
unless you add more enzyme
in a kinetic graph of VELOCITY vs concentration, what does the graph’s plateau/asymptote tell you about the substrate CONCENTRATION?
that it is saturated
Dr. Shimko called this ‘near infinite concentration of the substrate’
in enzyme kinetics for our class, why is the rxn rate called ‘initial velocity’ instead of just ‘velocity’?
‘initial velocity’ describes the rate of the reaction BEFORE equilibrium is achieved
in the context of Michaelis-Menten what does ‘steady state’ describe?
the point at which the [ES] is constant over time
rate of ES formation = rate of ES breakdown
or
rate of ES formation = rate of ES consumption
what units does the Km have?
units of concentration
when does Km = [substrate]?
when a reaction achieves half of its maximum velocity
what does the Michaelis-Menten eqn allow us to solve for?
you can use the terms to solve for the other terms (Km, Vmax, K2)
the allows you to examine the stability of the ES complex and the rate of product formation
how can you tell if a rxn rate graph is showing you one substrate at a single concentration or multiple substrates?
a graph of multiple substrates will have multiple lines rather than just a single line
what is the expression for Km?
Km = [E][S]/[ES] = k-1/k1
what does [E]_T represent?
total enzyme concentration
How is Km similar to Kd?
both communicate the degree of affinity between the bonding partners in question
both essentially show you rate or reverse rxn/rate of forward rxn
why do we exclude K2 from the numerator of the Km expression?
its associated with the rate limiting step but its actual value is negligible compared to the other term in the numerator (K-1) so we just ignore it under Michaelis-Menten conditions
How can K2 both be the rate limiting step but also be small enough to omit from the Km equation?
I will ask Milo
what are the main four conditions assumed to be present under Michalis-Menten conditions?
The rxn has a huge amount of substrate present (compared to the amount of enzyme)
Once the rxn gets going, the [ES] remains constant over time (aka the rxn is in a ‘steady state’)
Once the rxn achieves maximum velocity, all the enzymes are busy at work in their ES state; None of them are loafing around in their ‘free’ enzyme state.
As the rxn proceeds, the k2 (ES to E + P) step is the rate limiting step, and therefore, its rate law is used as the rate law for the entire rxn
what is the rate law expression for the k2 step ( ES to E + P] of a Michaelis-Menten rxn?
v_0 = k2[ES]
What is the significance of applying Michaelis-Menten approach to understanding rxns?
It allows us to describe reaction proceedings mathematically without having to account for every single variable impacting the rxn’s rate
why is the steady state approximation the basis for the entire Michaelis-Menten equation?
it gives the equation/mathematical relationship that the Michaelis-Menten equation is derived from.
Forms the basis for which the other math terms (like Vmax and Km) can be combined into a single, simple mathematical expression (the Michaelis-Menten eqn)
in a catalysis expression, where are the two places where ES is can be consumed?
- it can be consumed as it reverts to E + S from ES
2. it can be consumed as it converts from ES to E + P
in a catalysis expression, where is the one place where ES is can be produced?
it can be produced during the rxn’s ‘binding’ step that converts E + S to ES
what are the two basic rxn steps Michaelis-Menten used to summarize catalytic rxns?
- enzyme-substrate binding step
2. catalytic step
In the context of Michaelis-Menten summary of catalytic rxns, what happens in the step one?
reversible binding of E and S to create ES complex
In the context of Michaelis-Menten summary of catalytic rxns, what happens in the step two?
NOT reversible conversion of ES to E + P (where E releases the P)
what is the general expression for a catalyzed rxn as Michaelis-Menten described it?
E + S in equ. with ES goes to E + P
what is the expression for the Michaelis-Menten eqn?
V = (vmax * [S]) / (Km + [S])
what is the expression for the Vmax?
Vmax= k2 * [E]total
where K2 is the rate constant from the rxn’s slowest step and [E]total is the concentration of enzymes (being 100% in the ES state) at maximum velocity.
what two math maneuvers do you need to do to get from the Michaelis-Menten equation to the Lineweaver-Burk eqn?
- divide the Michaelis-Menten eqn by 1
2. do algebra to make the resulting eqn conform to the y-mx+b format
Does the plot for a Michaelis-Menten appear as a curve or a line?
a line then a curve
Does the plot for a Lineweaver-Burk appear as a curve or a line?
a line
What does the slope on (the linear part of) a Michaelis-Menten plot tell you?
initial velocity
What does the slope on Lineweaver-Burk plot tell you?
km/vmax
What does the y intercept on Lineweaver-Burk plot tell you?
1/vmax
What does the x intercept on Lineweaver-Burk plot tell you?
-1/Km
What does the y axis on Lineweaver-Burk plot tell you?
1/velocity
What does the x axis on Lineweaver-Burk plot tell you?
1/[substrate]
What does the y axis on Michaelis-Menten plot tell you?
velocity
What does the x axis on Michaelis-Menten plot tell you?
[substrate]
How would you quickly find the Vmax from the Lineweaver-Burk eqn?
1/y int
1/blue box
How would you quickly find the Km from the Lineweaver-Burk eqn?
slope/y int
red box/blue box
What are the 3 types of inhibitors we talk about in our class?
noncompetitive
UNcompetitive
competitive
which quantity does competitive inhibitors impact? vmax or Km?
competitive inhibitors increase km (because they decrease enzyme-substrate affinity
why are competitive inhibitors so called?
because they bind to the same active site as the substrate
they compete for binding
how/can you overcome the impact of a competitive inhibitor?
add a dramatically large amount of substrate
what is the general expression for how/where competitive inhibitors interfere with catalysis?
EI in equi. with I + E + S in equi. with ES goes to P + E
how does a competitive inhibitor impact a Michaelis-Menten plot?
it shifts the Km to the right
because it makes the Km bigger/enzyme-substrate affinity lower
how does a competitive inhibitor impact a Lineweaver-Burk plot?
it keeps the same y int but the slope is much steeper (because the km is in the numerator of the slope)
why are NON competitive inhibitors so called?
they bind to a different active site than the substrate
if NON competitive inhibitors don’t use the same binding site as substrates, how is it that they can inhibit catalysis?
when they bind to the other site, it changes the conformation of the substrate site, making it slower in its catalysis activity
which quantity does NONcompetitive inhibitors impact? vmax or Km?
vmax
what is the general expression for how/where NONcompetitive inhibitors interfere with catalysis?
see notes…
how does a NON competitive inhibitor impact a Michaelis-Menten plot?
Km stays the same but Vmax is smaller
how does a NON competitive inhibitor impact a Lineweaver-Burk plot?
x int stays the same but y int is higher and slope is steeper
When you look at the Lineweaver-Burk plot, which point(s) on the graph have the highest substrate concentration?
The points lowest on the graph (the closer you get to the origin, the higher the substrate concentrations are)
It looks backwards because it’s an inverted graph
What does the k_cat represent?
The k constant for the slowest (rate limiting) step in the catalysis
Most of them for our class have been the k2
What is the expression for the specificity constant?
Kcat/km
What does the specificity constant tell you?
It tells you how well a substrate and enzyme interact
Tells you how well an enzyme will act upon a given substrate
If a substrate has large specificity constant relative to a given enzyme, it means they interact well and make for a ‘productive’ enzyme-substrate pair
Do enzyme inhibitors have their own dissociation constants?
Yes
Why did Dr. Shimko tell us about the malonate inhibitor?
This is an example of competitive inhibition.
If malonate binds to succinate dehydrogenase enzyme (in the active site meant for succinate), the enzyme won’t be able to catalyze the transformation of succinate into fumarate.
What is an ‘alpha’ Km?
What you call it when an inhibitor causes a rxn km to increase.
What is the expression for the enzyme inhibitor’s dissociation constant?
Ki =[E][I] / [EI]
What is the difference between the M & M eqn for an UNinhibited rxn and the eqn for an inhibited rxn?
In the INhibited version, the k constant term is alphaKm instead of just Km
What is the expression for the ‘alpha’ in ‘alpha Km’?
1 + ([I] / Ki)
Will you have to calculate Ki or alphas for this class?
No
What kind of inhibitor is a transition state analog: competitive, non-competitive, un-competitive, catfishpetitive?
Competitive
What is a bisubstrate analog?
A competitive inhibitor molecule that can (simultaneously) bind to an enzyme in more than one of the enzyme’s active sites. This increase in interactions makes the inhibitor EXTRA able to block the enzyme from binding to the things it was supposed to bind to
Look at the REC assignment
Why did Dr. Shimko tell us about PALA?
It’s an example of the bisubstrate analog that he never explained.
how does an UNcompetitive inhibitor impact a Lineweaver-Burk plot?
Both the Km and the Vmax decrease, so the graph shifts to the left (for the Km), shifts up (for Vmax) and the slope is unchanged (because the alpha term cancels from ‘m’ term in the slope version of the Lineweaver-Burk equation)
What is a ‘mixed’ inhibitor?
An inhibitor that can bind both to the enzyme AND the ES complex
What distinguishes NONcompetitive inhibition from other kinds of mixed inhibition?
In NONcompetitive inhibition, the Ki = Ki’
What does it mean to say that NONcompetitive inhibition has a Ki = Ki’
It means the inhibitor is just as likely to bind with the enzyme as it is to bind with the ES complex
Why did Dr. Shimko show us the Iodoacetamide inhibitor?
It’s an example of an irreversible inhibitor that is bad because it inhibits enzymes indiscriminately, rendering them unable to catalyze rxns ever again
Why is the iodoacetamide drug good at doing irreversible inhibiting?
It has a halide on it that is very attractive to enzymes with nucleophilic residues (such as catalytic cysteine)
Why did Dr. Shimko show us the DIPF irreversible inhibitor?
It’s another example of an inhibitor that is bad because it is too permissive/not selective enough
It is somewhat selective in that it only binds to activated serine, but it will still bond to any activated serine
Why did Dr. Shimko show us the drug TPCK inhibitor?
It’s an example of a ‘reactive substrate analog’
Chpt 8 part 4 time stamp 1:12:40
Generally speaking, does an inhibitor replace the enzyme in catalysis or the substrate?
substrate
