Chapter 12 : Enzyme Kinetics, Inhibition, and Control Flashcards
What is enzyme kinetics? What have effects on enzymes? How is the activity of enzymes controlled? How are inhibitors practically used?
-The study of enzymatic reaction rates
-Inhibitors
-Allosteric effects and covalent modification which involves Protein Phosphorylation
-As drugs
*
* Derive the Michaelis–Menten equation.
- What do the values of K M and k cat /K M reveal about an enzyme?
- Why can’t an enzyme have a k cat /K M value greater than 10 9 M −1 ∙ s−1 ?
- Write the Lineweaver–Burk (doublereciprocal) equation and describe the features of a Lineweaver–Burk plot.
- Why can’t enzyme kinetics prove that a particular enzyme mechanism is correct?
- Use Cleland notation to describe Ordered and Random sequential reactions and a Ping Pong reaction.
Purpose of reaction orders? What are the 2 types of elementary (simple molecular processes) reaction orders? How do velocity and reaction concentration relate in the two and what are the units of velocity and k in each?
Reaction Orders Indicates the Number of Molecules Participating in an Elementary Reaction.
- First order reaction/Unimolecular = A → P,
Velocity is proportional to reactant concentration (A)
Velocity has units of molar per second (M · s−1)
K has units of s-1 - Second order reaction/Bimolecular = 2A → P & A + B → P
Velocity is proportional to the square of the concentration of A
Velocity has units of molar per second (M · s−1)
K has units of M −1· s−1
-1 just means your on the denominator of a reaction
What is velocity? What are the equations to determine velocity in both reaction orders?
-the instantaneous rate of appearance of product or disappearance of reactant (v) / how much P appears as a function of time
First order: v = d[P]/dt =- d[A]/dt = k[A]
Second order: v = -d[A]/dt = k[A]2
v = -d[A]/dt = -d[B]/dt = k[A] [B]
k =?
The proportionality constant is known as a rate constant
k =?
The proportionality constant is known as a rate constant
At constant temperature, the rate of an elementary reaction is proportional to ? Meaning?
the frequency with which the reacting molecules come together.
-the more our 2 molecules come together the faster the reaction
btw and rate constant, K, tells us the speed of the reaction
Purpose of Rate Equations?
Rate Equation Indicates the Progress of a Reaction as a Function of Time.
Purpose of Rate Equations?
Rate Equation Indicates the Progress of a Reaction as a Function of Time. They are derived from equations that describe instantaneous reaction velocity.
instantaneous reaction velocity
the rate of a reaction at any particular point in time
What is Half life? What are the equations? When is it constant?
-the time for half of the reactant to initially decompose/how long till Ao becomes half of its starting concentration
First order: t1/2= ln 2/k = 0.693/k
Second order: 1/[A]=1/[A]0 +kt
Constant for first order not second order
what is a pseudo-first-order reaction?
finding the rate constant of a second order reaction by increasing the concentration of one reactant, so the reaction rate only depends on lesser reactant. this makes the reaction appear to be in first order with respect to the reactant with the lower concentration.
This is due to the second order not being constant and depending on the intitial reactant concentration.
Enzyme Kinetics Often Follows the Michaelis–Menten Equation. Describe the general enzyme reaction.
What is the velocity of making product equation and change in ES over time equation?
-vo = V max [S]/K M + [S]
-Enzyme plus Substrate will form an Enzyme and Substrate complex with a rate constant of k1.
This can break down to reform the Enzyme plus Substrate with a reaction order/rate constant of k-1.
The ES can make product plus enzyme which has a rate constant of k2
Equations on page 365
To make an equation for enzyme kinetics, we need some assumptions, what are they? What is Ks?
- The second reaction is irreversible. Once we make product we cannot go back
- The amount of ES is not going to change, Its at a steady state. So change in ES over Time =0. At equilibrium?
- Reverse reaction of ES breaking down to E+S is much bigger than the k2 reaction that makes Product. Much more likely to break apart into E+S than to make product
k1>k2
Ks is reverse rate constant/dissociation rate constant of the first step
equation on page 365
Reactions need to have measurable quantities, what are those in terms of ES and E
The quantities [ES] and [E] are not, in general, directly measurable, but the total enzyme concentration is usually readily determined.
Michaelis constant symbol and equation? Symbol and Equation for initial velocity, max velocity equation, and ES?
KM
KM= k−1+k2/k1
Initital velocity = v0
eq on pg 366 and 367
What are the differences between instantaneous velocity, initial velocity, and maximal velocity for an enzymatic reaction?
Instantaneous velocity is a velocity at a given time,
initial velocity is velocity at t=0
maximal velocity is when the enzyme is fully saturated with substrate.occurs at high substrate concentrations when the enzyme is saturated, that is, when it is entirely in the ES form. all enzyme has substrate
What is the Michaelis-Menten equation?
vo = V max [S]
————
K M + [S]
the basic equation of enzyme kinetics.
when substrate =KM what happens to speed
1/2 max speed
what is KM?
substrate concentration at which the velocity is half-maximal.
Km gives an idea of how good of binding the enzyme is.
When is Km catalytically efficient?
at low substrate concentrations
-The smaller the km the faster or easier it is it get to v max,
-smallest Km means highest affinity, can bind substrate better at lower concentrations
What is Kcat? What is kcat/KM?
Catalytic constant. the turnover number of an enzyme because it is the number of reaction processes (turnovers) that each active site catalyzes per unit time/how many reactions per active site per time
the rate at which an enzyme converts its substrate
a Measure of Catalytic Efficiency
is a measure of how efficiently an enzyme converts substrates into products.
The bigger the number the more efficient enzyme is.
eq on pg 368
The name for the most efficient enzymes?
-Diffusion-controlled limited Kcat/kM of 10^8 to 10^9 M −1∙ s−1.
These enzymes catalyze a reaction almost every time they encounter a substrate molecule.
Meaning the slowest step of reaction is the substrate diffusing to the enzyme
How are values for Vmax and KM determined? What is the slope, x, and y intercept?
Kinetic Data Via Lineweaver -Burk (double reciprocal) plot
change substrate con, hold enzymes constant and measure initial velocity
slope = km/vmax
x=-1/kM
y=1/vmax
…Any enzyme u can find Km and vmax, once u find vmax u find kcat, once you know km and kcat, you can find catalytic efficiency
What is a flaw of steady state kinetics?
Unfortunately, steady state kinetic measurements are incapable of revealing the Reaction Mechanism such as number of intermediates or nature of ES in an enzyme-catalyzed reaction
unable to tell how reaction runs, can only see product not mechanism.
know what goes in and out but not in between
input pressure and output flow can be measured
steady state kinetic measurements can provide a phenomenological description of enzymatic behavior, but the nature of the intermediates remains indeterminate.The existence of intermediates must be verified independently, for example, by identifying them through the use of spectroscopic techniques.
the two mechanisms that could be used by enzymes?
Bisubstrate reactions: Sequential reactions
and Ping pong
Bisubstrate reactions?
Bi-using 2 substrates
Couple ways it could happen:
Sequential- substrates must combine with the enzyme before a reaction can occur and products be released
Two types:
Random- those with no preference for the order of substrate addition
Ordered-those with a compulsory order of substrate addition to the enzyme
In the Ordered mechanism, the binding of the first substrate is apparently required for the enzyme to form the binding site for the second substrate, whereas in the Random mechanism, both binding sites are present on the free enzyme.
Ping Pong
Group-transfer reactions in which one or more products are released before all substrates have been added
substrate on, product off
Ping Pong Reactions Occur via Double Displacements.
Can determine difference through advanced experiements
Given are the five Km values for the binding of substrates to a particular enzyme. Which has the strongest affinity when k1 is greater than k2? (explain why)
the smallest value of Michaelis Menten constant will lead to the strongest enzyme substrate affinity
smallest Km means highest affinity, can bind substrate better at lower concentrations
convert to M
mM10^-3= M
um10^-6=M
nM10^-9=M
pM10^-12=M
Many substances alter the activity of an enzyme by combining with it in a way that influences what?
the binding of substrate and/or its turnover number.
what is a inhibitor?
Substances that reduce an enzyme’s activity in this way are known as inhibitors.
–Some enzyme inhibitors are substances that structurally resemble their enzyme’s substrates but either do not react or react very slowly. These substances are commonly used to probe the chemical and conformational nature of an enzyme’s active site in an effort to elucidate the enzyme’s catalytic mechanism.
Other inhibitors affect catalytic activity without interfering with substrate binding. Many do both
what is a Reversible inhibitor? 3 types?
- Competitive- Involves Inhibitor Binding at an Enzyme’s Substrate Binding Site, and product inhibition. directly blocks the active site
competes with the substrate to bind with the active site.The competitive inhibitor has a close structural resemblance with the substrate. The inhibitor may combine with the enzyme (E), forming an enzyme-inhibitor (EI) complex instead of an enzyme-substrate (ES) complex. The degree of inhibition depends upon the concentrations of the substrate and the inhibitor.A competitive inhibitor diminishes the rate of the reaction by reducing the proportion of the enzyme molecules bound to a substrate. Takes more substrate to outcompete I.
During competitive inhibition, the enzyme is inactivated when an inhibitor is bound, whether or not substrate is also present.
2.Uncompetitive Inhibition- Involves Inhibitor Binding to the Enzyme–Substrate Complex. binding of inhibitors to the enzyme at a place other than the active site.Uncompetitive inhibition is the inhibition of enzymatic activity by the binding of the inhibitor at an allosteric site like in the case of noncompetitive inhibition but the binding takes place with the enzyme-substrate (ES) complex, and not the free enzyme molecule.
The mechanism of inhibition involved in uncompetitive inhibition is by the removal of an activated enzyme-substrate complex which causes a decrease in the maximum velocity of the chemical reaction.
3.Mixed/Noncompetitive Inhibition- Involves Inhibitor Binding to Both the Free Enzyme and the Enzyme–Substrate Complex
The tern noncompetitive suggests that there is no competition between the substrate and the inhibitor for the binding to the active site and also has no structural resemblance to the substrate. In noncompetitive inhibition, the inhibitor and substrate can simultaneously bind to the same enzyme molecule since their binding sites are different and also, do not overlap.The binding of the inhibitor brings about conformational changes in the active site of the enzyme, which prevents the binding of the substrate molecule. Inhibitor bind to the allosteric site, changes the shape of the active site.Besides, the inhibitor effectively lowers the concentration of active enzymes and hence lowers the rate of reaction.
Noncompetitive inhibition, unlike competitive inhibition, cannot be overcome by increasing substrate concentration
These inhibitors do not undergo chemical reactions but are easily removed by dilution or dialysis
What is an Inactivator? Give an example.
Irreversible enzyme inhibitors that bind to the enzyme so tightly that they permanently block the enzyme’s activity.
Reagents that chemically modify specific amino acid residues can act as inactivators.For example, the compounds used to identify the catalytic Ser and His residues of serine proteases (Section 11-5A) are inactivators of these enzymes.
What is an Inactivator? Give an example.
Irreversible enzyme inhibitors that bind to the enzyme so tightly that they permanently block the enzyme’s activity.
Reagents that chemically modify specific amino acid residues can act as inactivators.For example, the compounds used to identify the catalytic Ser and His residues of serine proteases (Section 11-5A) are inactivators of these enzymes.
Reversible enzyme inhibitors
diminish an enzyme’s activity by interacting reversibly with it.
What is the competitive inhibitor?
A substance that competes directly with a normal substrate for an enzyme’s substrate-binding site. Such an inhibitor usually resembles the substrate so that it specifically binds to the active site but differs from the substrate so that it cannot react as the substrate does.
What is product inhibition? . Product inhibition is one way in which the cell..?
When there’s so much product, it’ll bind the active site and stop the reaction
reaction stops because substrate cant bind because product is overwhelming the enzyme
In this phenomenon, a product of the reaction, which necessarily is able to bind to the enzyme’s active site, may accumulate and compete with substrate for binding to the enzyme in subsequent catalytic cycles. Product inhibition is one way in which the cell controls the activities of its enzymes
Product inhibition is a type of enzyme inhibition where the product of an enzyme reaction inhibits its production
Product inhibition can be Competitive, non-competitive or uncompetitive.
What are Transition state analogs? Why are these particularly effective?
Inhibitor,
This is because effective catalysis often depends on an enzyme’s ability to bind to and stabilize its reaction’s transition state (Section 11-3E). A compound that mimics a transition state exploits these binding interactions in ways that a substrate analog cannot.
-Because it mimics/looks like the transition state.
The Degree of Competitive Inhibition Varies with? Is Vmax or KM changed?
the Fraction of Enzyme That Has Bound Inhibitor.
no, except km that increases with inhibitor
A competitive inhibitor therefore reduces the concentration of free enzyme available for substrate binding.
What is Ki? The smaller the number? How too determine Ki
The inhibition constant
the smaller the number the stronger the binding
The more inhibitor the more KM because we need substrate to compete with inhibiotn
Using a double reciprocal plot
uncompetitive inhibition’s line weaver Burk forms. What does this do to the active site? Are Vmax and KM affected? does adding substrate reverse inhibition?
parallel lines
The binding of uncompetitive inhibitor, which need not resemble substrate, presumably distorts the active site, thereby rendering the enzyme catalytically inactive.
yes both are lowered
-no Uncompetitive inhibition requires that the inhibitor affect the catalytic function of the enzyme but not its substrate binding.substrate decreases
Mixed(Noncompetitive) inhibition. Where does it bind? Are Vmax and KM affected
To both substrate binding and catalysis (metal ions)
Vmax decreases, Km can either increase,decrease, or stay the same
Lines crossing at a point not on any axis’
Draw the graphs representing Mixed(Noncompetitive) inhibition, competitive inhibition, and uncompetitive
What is pure noncompetitive inhibition.
When the inhibitor binds to both the free enzyme and the enzyme-substrate complex with equal affinity and only Vmax is affected by decreases and KM is unchanged.
what are the two major ways to control catalytic activity?
- control enzyme availability by increasing or decreases the rate of synthesiz and degradation of enzymes
- Control of enzyme activity by modifying their catalytic activity via inhibitors, allosteric mechanisms/modulation via structure alterations
what are the two major ways to control catalytic activity?
- control enzyme availability by increasing or decreases the rate of synthesiz and degradation of enzymes
- Control of enzyme activity by modifying their catalytic activity via inhibitors, allosteric mechanisms/modulation via structure alterations
Allosteric meaning?
means your binding something that is affecting catalytic mechanisms to speed up or slow it down, through structural alterations that influence the enzyme’s substrate-binding affinity or turnover number
Doesn’t bind to active site
ATCase is allosterically inhibited by cytidine triphosphate (CTP), a pyrimidine nucleotide, and is allosterically activated by adenosine triphosphate (ATP), a purine nucleotide. meaning
Also describe graph
CTP slows it down while ATP speeds it up
binding curve on graph is sigmoidal rather than hyperbolic.This is consistent with cooperative substrate binding like hemoglobin’s O2-binding curve is also sigmoidal;atc ase has multiple binding sites mutliptle products being made at one time
ATCase given ATP curve shifts where?
Left-speeds up. Takes less substrate to go faster
ATCase given CTP curve shifts where?
Right-goes slower. Needs more substrate to go faster
Why does CTP slow down the reaction
is an example of a feedback inhibitor, since it inhibits an earlier step in its own biosynthesis
when cap is high it binds to ATCase and inhibit CTP production until ATP=CTP
ATCase has two states what are they?
T State = Inactive
R State = Active
ATP binds to R state
CTP binds to T state
Substrate analog binds to R but not T
if CTP binds to r state, create large changes shifting to t state
if ATP binds to t state, shifts to r state, increasing catalytic activity
Binding causes what?
conformational change, causes tertiary and quaternary shifts
Most enzymes are modified by what
covalent modification
What is covalent modifcation? What amino acids are Phosphorylated in ATCase
Phosphorylation and Dephosphorylation,
mostly Phosphorylation (the attachment and removal of a phosphoryl group)
of the hydroxyl group of a Serine, threonine and tyrosine
The enzymes that catalyze the phosphorylation and dephosphorylation of glycogen phosphorylase during Covalent Modification are called
protein kinases and protein phosphatases, alter the activities of the modified proteins.
what are isozymes
catalytically and structurally similar but genetically distinct enzymes from the same organism;
What Are Often Implicated in Adverse Drug Reactions
Cytochromes P450
Drug Discovery Employs a Variety of Techniques like what
Structure-Based Drug Design Accelerates Drug Discovery.
Combinatorial Chemistry and High-Throughput Screening Are Useful Drug Discovery Tools.
Drug Discovery Employs a Variety of Techniques like what
Structure-Based Drug Design Accelerates Drug Discovery.- uses structure to guide development
Combinatorial Chemistry and High-Throughput Screening Are Useful Drug Discovery Tools.
using technology to synthesize compounds rapidly, cheap, combined with the robotic high throughput screening to make test many compounds
A Drug’s Bioavailability Depends on ?
How It Is Absorbed and Transported in the Body
A Drug’s Bioavailability Depends on ?
How It Is Absorbed and Transported in the Body
A drug candidate that exhibits a desired effect is called a
lead compound
