Quiz 6 (Lectures 16 - 18) Flashcards
What are bisubstrate reactions?
Two substrates going to two products:
S1 + S2 -> P1 + P2
What are transferase reactions?
S1-X + S2 -> P1 + P2-X
X group is transferred
What are the two types of bisubstrate transferase reactions?
Sequential and ping pong
What happens in a sequential reaction?
Both substrates bind to form a ternary complex with the enzyme before any reaction occurs and any products are released.
What are the two types of sequential reaction mechanism?
- Ordered: Substrates are required to bind in a specific order
- Random: Either substrate can bind first
Enzymes that require NAD(P)+ as an oxidant often follow a(n) ______ mechanism.
ordered
In NAD(P)+ dehydrogenases, the oxidized coenzyme binds [first/second] (S1).
first
In sequential bisubstrate reaction, where is the point in which a ternary complex is formed on a double-reciprocal plot?
Where the lines all intersect
Describe the ping and the pong in a ping pong bisubstrate reaction.
- Ping: S1 binds and modifies the enzyme (E’) converting S1 to product (P1), which then leaves
- Pong: The following substrate (S2) binds, is modified by the enzyme to product (P2) and leaves
What’s the catalytic cycle?
In a ping pong bisubstrate reaction, the enzyme is regenerated during this process to complete the catalytic cycle (E’ -> E)
(Also known as double-displacement)
Ping pong bisubstrate reactions are characterized by ______ lines on a double-reciprocal plot. Why is this?
parallel
No ternary complex is formed during catalytic turnover.
The majority of drugs are _______.
inhibitors
How can inhibitor drugs inhibit the catalytic activity of an enzyme?
By binding to it
They block signaling pathways by binding to receptors or by binding to a specific protein kinase or phosphatase for a particular pathway.
What are four properties of drugs?
- Drugs need to be as specific as possible
- Drugs must be soluble
- Drugs must have a biological life-time that allows them to reach their target, but not too long that they aren’t eventually cleared from the body
- Drugs must be able to reach the cellular compartment or tissue where their target resides
What are the types of inhibition?
- Competitive (Transition state inhibitors)
- Uncompetitive
- Mixed (Special case: noncompetitive)
- Irreversible inhibitors (Special case: suicide)
In competitive inhibition, a competitive inhibitor competes for what?
The same binding site as the substrate
A competitive inhibitor often chemically resembles the _______.
substrate
Can the enzyme catalyze the competitive inhibitor’s conversion to anything else?
No
The best competitive inhibitors are similar to the ___________.
transition state
The binding sites of enzymes are optimized for binding the _______, which lowers the energy required to cross the _______ barrier.
transition state; transition state
When the substrate binds, the enzyme is set up to “encourage” the substrate to optimize its interactions with the enzyme even more by adopting the structure of the __________.
transition state
What’s the dissociation constant for inhibitor I?
KI = [E][I]/[EI]
On a double reciprocal plot, the data for a competitive inhibitor will produce a straight line that still crosses the _-axis at the same place as the data without the inhibitor.
y
(This is because when [S]»_space; [I], all active sites will be occupied by substrate, not inhibitor, leaving Vmax unaltered.)
The presence of a competitive inhibitor increases the experimentally measured ___, but doesn’t affect the ___.
Km; Vmax
An uncompetitive inhibitor can only bind to the ________.
ES complex
Uncompetitive inhibtion is [reversible/irreversible].
reversible
When does uncompetitive inhibition most commonly occur? How?
Multisubstrate reactions
The addition of substrates is controlled sequentially (ordered) by a conformational change induced by the binding of the first substrate that enables binding of the second substrate at a separate site.
An uncompetitive inhibitor often resembles the _________, but the enzyme cannot catalyze its reaction with the ________.
second subtrate; first substrate
The presence of an uncompetitive inhibitor decreases the experimentally measured ___ and ___ from their “true” values.
Vmax; Km
On a double reciprocal plot, uncompetitive inhibition gives _____ lines for different ___.
parallel; [I]
What does a mixed inhibitor do?
Binds at other site on the enzyme away from the substrate binding site
Is mixed inhibition reversible or irreversible?
Reversible
A mixed inhibitor can bind to both the free enzyme and the ES complex. True or false?
True
The binding of a mixed inhibitor can alter the binding affinity of the ______ and _______ for the enzyme via a conformational change.
substrate; inhibitor
On a double reciprocal plot, the data for a mixed inhibitor will produce a ______ line.
The Km will be [true/apparent], and the Vmax will be [true/apparent].
straight
apparent; apparent
A mixed inhibitor can increase Km,app if what?
If I has more affinity for E over ES
A mixed inhibitor can decrease Km,app if what?
If I has more affinity for ES over E
A mixed inhibitor decreases ____.
Vmax
How does noncompetitive inhibition work?
A noncompetitive inhibitor binds at another site on the enzyme away from the substrate binding site.
It can bind to both the free enzyme and the ES complex.
The binding of the noncompetitive inhibitor [doesn’t/does] alter the binding affinity of the substrate for the enzyme. What does this mean?
doesn’t
This means there will be no conformational change. The substrate can still bind to the enzyme in the presence of the inhibitor, but the reaction will not proceed to product with inhibitor bound.
On a double reciprocal plot, the data for a noncompetitive inhibitor will produce a straight line that what?
Crosses the x-axis at the same place as the kinetic data without the inhibitor
The Vmax for an enzyme with a noncompetitive inhibitor will be [true/apparent].
apparent
The presence of a noncompetitive inhibitor ______ the Vmax, but ______ the Km.
decreases; doesn’t affect
Apparent Km equals the [S] when V0 is what?
0.5 apparent Vmax
What can a irreversible inhibitor do?
Bind covalently, destroy a key functional group, or form a very tight non-covalent complex
What does a suicide/mechanism based inhibitor do?
A suicide inhibitor undergoes the first few steps of the normal enzymatic reaction, but instead of forming the product, it forms a very reactive compound that combines irreversibly with the enzyme.
These inhibitors hijack the normal enzymatic reaction to inactivate the enzyme.
Name the seven ways proteins can be regulated.
- Allosteric regulation (Feedback inhibition)
- Covalent modification (Phosphorylation)
- Cellular compartmentalization
- Zymogen activation (Zymogens are inactive precursor forms of the enzyme)
- Protein synthesis/gene regulation
- Protein degradation
Protein synthesis and degradation has a much _____ response and produces _____-term effects.
slower; longer
What are the fast response types of protein regulation? What are these used in?
Allosteric, covalent modification, and zymogen activation
They’re used in signal transduction and enzyme pathways.
In vivo, intermediate products in a pathway don’t build up, so the flux through the pathway must be _____ at each enzyme catalyzed step.
the same
In vivo, the flux through the pathway is generally many times ____ than the in vitro measured capabilities of the component enzymes.
slower
Good candidate enzymes for regulation are at the [start/end/middle] of pathways.
start
What makes an enzyme a good candidate for regulation?
Enzymes where the forward reaction is highly favored over the back reaction
For most enzymes, their forward and backward reactions have _____ rates (reversible), and their overall reaction is close to the ____________ of the cell.
similar; dynamic steady state
Most enzymes aren’t good candidates for regulation. Why?
Their overall reaction is close to the dynamic steady state of the cell. An increase in flux in the forward direction is countered by an equal increase in the backward direction, and a steady state is reestablished.
Changes in specific enzyme activity require which three forms of protein regulation?
- Allosteric regulation
- Covalent modification
- Zymogen activation
Changes in levels of enzymes are controlled by which two forms of protein regulation?
- Gene regulation/protein synthesis
- Protein degradation
Regulatory enzymes generally have [single/multiple] subunit(s).
multiple
What’s an allosteric modulator?
Substrate in which the allosteric site and the active site are the same
(Homotrophic allosteric modulation)
In metabolism, most modulators are ______, meaning that they bind to sites separate from the active site, inducing a conformational change.
heterotrophic
Molecules that enhance the catalytic activity are called…
allosteric activators
Allosteric activators _____ the Km and/or _____ the Vmax.
decrease; increase
Molecules that decrease the catalytic activity are called…
allosteric inhibitors
Allosteric inhibitors _____ the Km and/or _____ the Vmax.
increase; decrease
The action of allosteric modulators leads to _____ kinetics, deviating from Michaelis-Menten behavior.
sigmoidal
Regarding allosteric enzymes, the [S] at 0.5Vmax is ____.
K0.5
What is variable is commonly altered with a heterotrophic modulator? Less commonly altered?
Common: K05 is altered, not Vmax
Less common: Vmax is altered, not K0.5
Substrate can still bind to the T state, but with ____ affinity.
lower
How does negative regulation occur?
A late product from a multienzyme pathway binds to an allosteric site (regulatory site) on an early acting enzyme in the pathway and inhibits its action
How does positive regulation occur?
The product from one pathway stimulates an early enzyme in a different pathway
The allosteric and active binding sites are linked how?
The binding to either site affects the ability to bind at the other site
If two ligands bind to the same conformation of the protein, one ______ the binding of the other, then _____ occurs.
enhancing; activation
If two ligands bind to different conformation of the protein, one _____ binding of the other through a conformational switch, then _____ occurs.
reducing; inhibition
The presence of a large amount of substrate will [stabilize/destabilize] the active form.
This _____ the binding affinity for the allosteric inhibitor (late product), which stabilizes a _____ activity form of the enzyme.
stabilize
reduces; lower
What’s catabolism?
The breakdown of energy containing nutrients to energy-depleted products
What’s anabolism?
The synthesis of more complex molecules from simpler ones, which normally requires free energy
The cell is in a high energy state if there is an abundance of what three compounds?
- ATP
- Acetyl CoA
- Citrate
The cell is in a low-energy state if there is an abundance of what two compounds?
- ADP
- AMP
Catabolism pathways are inhibited by ____ and activated by ____ and ____.
ATP; ADP; AMP
Anabolic pathways are activated by ____ and inhibited by ____ and _____.
ATP; ADP; AMP
Phosphorylation of Ser, Thr, or Thy residues in proteins is used to do what?
Reversibly regulate protein function through conformational changes or to enable binding events in signal transduction
At any time in a eukaryotic cell, about ____ of all proteins are phosphorylated.
1/3
Enzymes that phosphorylate are called _______, and enzymes that dephosphorylate are called _______.
protein kinases; protein phosphatases
Phosphorylation can ______ or ______ the enzyme.
switch on; switch off
Enzymes that are produced as inactive forms are called what?
Proenzymes or zymogens
What are the three ways that proenzymes/zymogens are activated?
- Proteolytic cleavage
- Post-translational modification
- Cofactor insertion
Proteases are inactivated by ______ proteins that bind very tightly to the enzyme active site.
inhibitor
What’s a proteasome? What does it consist of?
Efficient cellular machinery that degrades proteins
It consists of a 20S core and two 19S caps
What does ubiquitin do?
Proteins targeted to the proteasome by the covalent attachment of multiple copies of ubiquitin
How does ubiquitin attach to proteins?
Ubiquitin’s C-terminus is covalently linked to Lys on side-chains in an isopeptidic bond, both in target proteins and also to itself
Ubiquitin-conjugating enzyme (E2) forms a complex with ubiquitin ligase (E3). True or false?
True
What’s E1?
Ubiquitin-activating enzyme
How does E1 activate ubiquitin?
By forming a high energy thioester bond with the C-terminus and an E1 Cys residue (Needs ATP)
E1 transfers the ubiquitin to a ___ residue on an activated E2-E3 complex.
Cys
E3 recognizes a specific degradation signal on the target protein, enabling E2 to do what?
Transfer its ubiquitin to a Lys on the target
Multiple rounds of ubiquitin charged E2-E3 binding lead to the formation of what?
A multiubiquitin chain attached to the target (Minimum length of 4)
(Recognized by the 19S cap of the proteasome)
The N-terminal residue of proteins can target the protein for degradation. The identity of the N-terminal amino acid correlates to what?
The half-life of the protein in the cell
Certain amino acids will lead to a protein being tagged for degradation more frequently than others, creating a _____.
“clock”
Describe specific acid-base catalysis.
The transfer of a proton can stabilize the unstable charged intermediate to form a species that more readily breaks down to products rather than reactants
The intermediate is stabilized only in what conditions?
If protons can be transferred between water and the intermediate more rapidly than the breakdown to reactants
True or false? The presence of additional H+ donors/acceptors have an effect on the reaction rate.
False. They have no effect on the rate.
What is general acid-base catalysis?
Proton transfers mediated by other groups, such as amino acid side-chains
In general acid-base catalysis, are all the intermediates that are formed stabilized?
No, only a fraction
In general acid-base catalysis, the presence of alternative proton donors/acceptors [increases/decreases/doesn’t change] the rate of the reaction.
increases
Ionic interactions with the enzyme-bound metal can do what?
Help orient a substrate correctly in the active site or stabilize a charged intermediate or transition state
Metals can also mediate what kind of reactions?
Oxidation-reduction reactions
Nearly ____ of all enzymes require metal(s) for catalytic activity.
one third
ATP4- and ADP3- always bind to enzymes as a complex with ____.
Mg2+
(This shields the negative charges of the terminal phosphate, making it an easier target for nucleophilic attack)
H2O can easily enter the hexokinase active site, but the enzyme favors the reaction with glucose by 10^6. How does the enzyme discriminate between glucose and H2O?
The enzyme undergoes a conformational change upon substrate binding (induced fit)
When glucose is not present, the enzyme is inactive. How does it become active?
The binding energy derived from gluclose and MgATP2- binding induces the conformational change to the active catalytic form (Blocks H2O access and brings glucose and MgATP2- closer)
When xylose binds to hexokinase instead of glucose, what happens?
Xylose can’t be phosphorylated, but the binding energy is close enough to that of glucose that hexokinase undergoes a conformational change to the active form.
H2O is the major substrate that is phosphorylated.
Aside from induced fit, the hexokinase mechanism also requires what three things?
- General acid-base catalysis
- Metal ion catalysis (Mg2+ bound to ATP)
- Transition state stabilization
Describe metal ion catalysis using enolase.
- First step: Lys345 acts as a general base catalyst removing a proton from C2
- In the enolase active site, the 2-phosphoglycerate has strong ionic interactions with two Mg2+
- This makes the C2 proton more acidic and easier to remove
- Other amino acids contribute hydrogen bonds that stabilize both the enolic intermediate and the transition state preceding it
- The magnesium ions also promote the formation and stabilization of the negative charges of the enolic intermediate
- Second step: Glu211 acts as a general acid catalyst, donating a proton to the hydroxyl leaving group producing water
What do proteases do?
Catalyze the hydrolysis of polypeptide backbones to yield smaller peptides
Why are serine proteases important?
They have a reactive Ser residue that’s essential for catalytic activity
Serine proteases are also what?
Esterases
Amide and ester bonds are ______.
isosteric (stereochemical analogs)
Why are amide bonds harder to cleave than ester bonds?
Amide bonds have a partial double bond character
Describe the esterase activity of serine proteases.
- Burst phase: Formation of an acyl-enzyme intermediate and release of the first product, p=nph (covalent catalysis)
- Steady state pnase: Slower hydrolysis of acyl-enzyme intermediate to release the second product, acetate
(Ping pong mechanism)
What are the two essential residues in chymotrypsin for activity? What’s the third invariant residue?
These three residues form a what?
Two essential residues: Ser195 and His57
The third: Asp102 (Buried in a solvent-inaccessible pocket)
They form a catalytic traid.
Chymotrypsin cleaves after ______.
Trypsin cleaves after _______.
Elastase cleaves after _______.
- bulky hydrophobics
- positively charged residues
- small neutral residues
What’s the serine protease mechanism?
- Nucleophilic attack by Ser OH
- Protonation of peptide N
- Deprotonation of H2O and attack carbonyl carbon
- Cleavage or Ser O-peptide bond
Analysis of serine proteases reveals an oxyanion hole. What does the oxyanion hole enable?
The preferential binding to the transition state
