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267 Cards in this Set

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  • Back
Enzymes can be __ or __
Proteins or RNA
In one sentence, how to enzymes catalyze reactions?
Enzymes catalyze reactions by stabilizing transition states.
What is a preolytic enzyme?
An enzyme that hydrolyzes (breaks by addition of water) a peptide bond
What is an enzyme that hydrolyzes a peptide bond called?
A proteolytic enzyme
What do proteolytic enzymes do in vitro?
hydrolysis of an ester bond.
What does trypsin do as an enzyme?
Hydrolyzes peptide bond on the carboxyl side of Arg or Lys.
What enzyme would you use to hydrolyze the peptide bond on the carboxyl side of an arginine or lysine?
Trypsin
What determines the specificity of an enzyme?
The interactions of substrate with enzyme, as a result of 3D structure of enzyme protein.
What is an apoenzyme?
An enzyme that requires a cofactor to be active.
What is a holoenzyme?
An active enzyme: apoenzyme + cofactor
What is the name of an inactive enzyme that requires a cofactor to be active?
Apoenzyme
What is the name of an active enzyme which has a bound cofactor?
Haloenzyme
What are the two types of cofactors?
1. metals
2. mall organic molecules
From what are most enzymes derived?
Vitamins
What are tightly bound cofactors called?
Prosthetic Groups
What are small organic molecules that act as cofactors called?
Coenzymes
What are coenzymes?
Small organic molecules that act as cofactors (activating apoenzyme). Usually loosely associated with enzyme; can be used by a variety of enzymes.
How are enzymes that use the same coenzyme similar?
They usually perform catalysis by similar mechanisms
How can we tell if a reaction will occur spontaneously?
If ∆G is negative = exergonic
What does exergonic mean?
∆G is negative, and the reaction can occur spontaneously
What is ∆G if a system is at equilibrium?
Zero -- no net change can take place
What does it mean if ∆G = 0?
The system is at equilibrium and no net change can take place
What is ∆G if a reaction cannot occur spontaneously?
Positive = endergonic
What does it mean if ∆ is positive?
The reaction can't occur spontaneously, it is said to be endergonic
What does the ∆G of a reaction depend on?
The free energy of the products (final state) minus the free energy of the reactants (initial state). Does not depend on the mechanism of reaction
Does the mechanism of the reaction have an effect on the ∆G (whether the reaction can occur spontaneously)?
No, only depends on free energy of products and reactants
Does ∆G tell us about the rate of reaction?
No. Tells us whether reaction can occur spontaneously, but not whether it is at a realistic rate.
What does the free energy of activation tell us?
The rate of reaction. Largely unrelated to the ∆G of the reaction.
What is the equation to determine ∆G?
∆G = ∆G⁰ + RTln[C][D]/[A][B]
What does the formula:
∆G⁰ + RTln[C][D]/[A][B]
tell us?
∆G; whether the reaction can occur spontaneously; if it is negative (exergonic), yes; if it is positive (endergonic), no.
What is ∆G⁰?
The free energy change for a reaction under standard conditions
What are "standard conditions" in biochemistry?
* reactants are present at 1M concentration
* pH=7.0
What is ∆G⁰' mean?
Standard conditions:
* pH = 7.0
* [H+] = 1.0
activity of water = 1
Do the (+/-) values of ∆G and ∆G⁰' (at pH 7) have to be the same?
No. Whether ∆G is larger, smaller or same as ∆G⁰ depends on concentration of reactants and products
How can we make a reaction spontaneous if ∆G⁰' is positive?
Alter the concentration of the products and reactants.
Does ∆G or ∆G⁰' determine whether reaction is spontaneous?
∆G
What can enzymes alter?
Can alter reaction rate.
NOT reaction equilibrium.
What does K=[P]/[S] tell us?
K = equilibrium constant.
P = product
S = substrate.
How do enzymes accelerate reactions?
They facilitate the formation of transition states. They decrease the activation energy.
What does a double dagger denote?
Transition state
What is "activation energy"?
The difference in free energy between transition state and substrate. Also known as Gibbs free energy of activation.
What is ∆G double dagger?
Gibbs free energy = activation energy
What does it mean that K=[P]/[S]=100?
The equilibrium concentration of P is 100 times greater than S
Do enzymes shift equilibrium positions?
No
How do you write that the equilibrium concentration of P is 100 times greater than of S?
K = [P]/[S] = 100
What accounts for the equilibrium of a reaction?
The free energy of reactants and products
How can we explain the rate enhancement in terms of thermodynamics?
Must consider reaction mechanism (pathway).
What evidence is there that enzymes complex with substrates?
1. at a constant concentration of enzyme, the rxn rate increases with increasing substrate until max velocity is reached.

2. x-ray crystallography (specifically, time-resolved crystallography)

3. spectroscopic characteristics of enzymes and substrates change when forming ES complex
What is time-resolved crystallography?
Depends on crystallizing a light-responsive (photolabile) substrate analog along with the enzyme. Exposure to a pulse of light converts the substrate analog into substrate, and image of the enzyme-substrate complex are obtained by scanning the crytsal with intense polychromatic x-rays
What is a good way of telling whether substrate and enzyme have formed a complex?
Look at spectroscopic characteristics. For example, enzyme alone and substrate along flouress at different wavelengths from enzyme-substrate complex.
What are teh five common features about enzyme active sites?
1. It is a 3D cleft/crevice
2. Takes up a relatively small part of the total volume of an enzyme
3. It is a unique microenvironment
4. It binds to substrates by multiple weak interactions
5. Its specificity of binding depends on defined arrangement of the atoms
What do we mean when we say that active sites are unique microenvironments?
Although stability is important, activity is more important. The microenvirnoment of the active cleft is usually nonpolar, but may contain polar residues to help in substrate binding.
What are the attractions between enzyme and substrate?
Weak, noncovalent attractions such as hydrogen bonds or van der Waals.
What are the strengths of enzyme-substrate binding?
-13 to -50kJmol⁻'
What are two models of enzyme-substrate binding?
1. lock-and-key model
2. induced fit model
What is the binding energy?
The free energy released when enzyme and substrate binds
What is the free energy released upon binding called?
binding energy
When is the maximal binding energy released?
When the enzyme facilitates the formation of the transition state
What accounts for specificity in enzyme-substrate binding?
The binding energy released - maximal binding energy is released when full complement of intereaction is formed, this happens when the substrate is in the transition state.
What does it do when enzyme binds to transition state substrate?
Lowers activation energy
What is the most stable interaction between enzyme and substrate?
Enzyme-Transition state (paradoxicaly, since this is the least stable reaction intermediate)
What determines whether enzyme-transition state complex will collapse into substrate or product?
The energy difference between the substrate and the product = ΔG
What is kinetics?
The study of reaction rates
What is the study of reaction rates called?
Kinetics
What is the study of rates of enzyme-catalyzed reactions called?
Enzyme Kinetics
What is enzyme kinetics?
The study of the rates of enzyme-catalyzed reactions
What is "V"
V = -ΔA/ΔT = ΔP/ΔT
= the quantity of A that disappears in a specified unti of time. It is equal to the rate of teh appearance of P (the quantity of P that appears in a specified unit of time)
What does the equation:
V=-ΔA/ΔT tell us?
The quantity of A that disappears in a specified unit of time
What is κ?
The rate constant.
The rate of rxn is directly related to the concentration of A by the rate constant.
What is a first-order reaction?
Reactions that are directly proportional to the reactant concentration. Take on the equation:
V=κ[A]
What is a reaction called if it is directly proportional to the reactant concentration?
First Order Reaction
What is the equation of a first order reaction?
V=κ[A]
What is a bimolecular reaction?
A reaction where there are two reactants.
What is the rate equation of a second order (bimolecular) reaction?
V=κ[A]^2

and

V=κ[A][B]
What are the units of second-order rate constants?
M⁻'s⁻'
What are pseudo-first order reactions?
second order reactions that appear to be first order, for example, if B is present in excess of A, rate will not appear to depend on [B]
What determines the extent of product formation?
Function of time for a series of substrate concentrations. Generally will increase with time, but at a certain point there will be no net change in the concentration of S or P
What is the rate of catalysis V₀?
the number of moles of product formed per second when the reaction is just beginning (t=0)
With many enzymes, how does V₀ vary?
It varies with substrate concentration.
What can the Michaelis-Menten equation tell us?
Model to account for kinetic characteristics.
What is the Michaelis-Menten Equation?
Michaelis-Menten
Who suggested the "steady state" assumption?
George Briggs and John Haldane
What is the steady-state assumption?
In a steady state, the concentration of intermediates (ES-complex) stay the same even if the concentrations of starting materials and products are changing.
When does the steady state occur?
When the rates of formation and breakdown of ES complex are equal
What is the equation of the steady state?
K₁[E][S]=(K₋₁ + K₂)/K₁
What does the steady-state assumption help simplify?
Combining the equation for rate of formation and rate of breakdown by assuming that in the steady state, [ES] stays the same even if the concentrations of starting materials and products are changing
What is the Michaelis-Menten equation?
Michaelis-Menten Equation
What does the steady-state assumption help simplify?
Combining the equation for rate of formation and rate of breakdown by assuming that in the steady state, [ES] stays the same even if the concentrations of starting materials and products are changing
What does the graph of Michaelis-Menten look like?
Michaelis-Menten
What does the steady-state assumption help simplify?
Combining the equation for rate of formation and rate of breakdown by assuming that in the steady state, [ES] stays the same even if the concentrations of starting materials and products are changing
What is the Michaelis-Menten equation?
Michaelis-Menten
What does the Michaelis-Menten Equation tell us when there is a very low substrate concentration?
When the [S] is much less than Km, V₀=Vmax/Km). What this means, is that the reaction is first order - the rate is directly proportional to the [S]. When [S] is much greater, the reaction order is zero - independent of [S]
What does it mean if the reaction is zero order?
That [S] is so high, that the rate of reaction is independent of its concentration
When [S] is extremely high, what is the order of the reaction?
Zero order
What does it mean when [S]=Km?
When [S] and Km are equal, then Km is equal to the substrate concentration at which the reaction is half its maximal value.
What is the [S] when Km and [S] are the same?
When [S] and Km are equal, then Km is equal to the substrate concentration at which the reaction is half its maximal value.
If an enzyme has a very high Km, what does this mean?
The enzyme achieves a high rate of catalysis only at very high concentrations of substrate.
What is Km?
The Michaelis-Menten constant. It is the concentration of substrate at Vmax/2.
What is the normal range that Km values lie in?
10⁻'M and 10⁻⁷M
What do Km values for a particular enzyme depend on? (4 things)
1. The particular substrate
2. pH
3. tempterature
4. ionic stregth
What is the Lineweaver-Burk (=double-plot) plot?
A plot of 1/V₀ versus 1/[S]. It gives us a straight line with:
* y-intercept = 1/Vmax
* x-intercept = -1/Km
* slope = Km/Vmax
What is the y-intercept of the Lineweaver-Burk plot?
1/Vmax
What is the x-intercept of the Lineweaver-Burk plot?
-1/Km
What is the slope of the Lineweaver-Burk plot?
Km/Vmax
Besides being the concentration of substrate at which half the active sites are filled, what is another meaning of the Km?
It is related to the rate constants of the individual steps in a reaction. Km is equal to the dissociation ocnstant of the ES complex if k₂ is much smaller than k₋₁
What is Km if k₂ is much smaller than k₋₁?
Under these conditions, Km is a measure of the strength of the ES complex
What does a high Km tell us with respect to the strength of the ES complex?
weak binding interaction of enzyme-substrate complex
What does a low Km tell us with respect to the strength of interaction of enzyme-substrate complex?
A low Km means that the substrate-enzyme complex has strong binding
When does Km indicate the affinity of the ES complex?
Only when k₋₁ is much greater than k₂
What is fES?
The fraction of active sites filled
What is the "symbol" for fraction of active sites filled?
fES
How are fES, Vmax, and Km related?
fES = V/Vmax = [S]/[S]+Km
Under physiological conditions, what is the [S]/Km ratio, typically?
0.01 and 0.1
What does the rate of catalysis equal when [S] is much greater than Km?
Vmax
When Vmax = [S], is [S] bigger or smaller than Km?
[S] is much greater than Km when the rate of catalysis = Vmax
What is the concentration of free enzyme when [S]<<Km?
The concentration of free enzyme, [E], is nearly equal to the total concentration of enzyme [E]t
What does the enzymatic velocity depend on when [S]<<Km?
The enzymatic velocity depends on kcat/Km, [S], [E]t when [S]<<Km
How can we compare an enzymes preference for a particular substrate (kcat) and the strength of enzyme-substrate complex?
Using kcat/Km values
How do we determine how efficient an enzyme can be?
Determine whether there are any physical limits on the value of kcat/Km.
What is the Circe Effect?
The attractive electrostatic forces that lure a substrate into an enzyme's active site
What are the attractive forces called that lure a substrate into an enzyme's active site?
The Circe Effect
What is a sequential reaction?
A reaction where all substrates bind to the enzyme before any product is released.
What type of reaction do most enzymes with NAD+ or NADH as a substrate undergo?
Sequential Reaction
What are the two types of sequential reactions?
Ordered and Random
What happens in an ordered sequential reaction?
Enzyme exists as a ternary complex consisting of
1st) enzyme and substrate
** catalysis**
2nd) enzyme and product
What happens in a random sequential mechanism?
The order of the addition of substrates and release of products is random. Still passes through the ternary complexes including
1st) substrates and
2nd) products
What is a double-displacement reaction?
One or more products are released before all substrates bind the enzyme
What is the defining feature of a double-displacement reaction?
The existence of a substituted enzyme intermediate in which the enzyme is temporarily modified; example: one substrate binds, then one product is released, then another substrate binds, then final product is released
Do allosteric enzymes obey Michaelis-Menten kinetics?
No
What type of enzyme does not obey Michaelis-Menten kinetics?
Allosteric Enzymes
What do the plots of allosteric enzymes look like?
Often, sigmoidal
Do allosteric enzymes obey Michaelis-Menten kinetics?
No
What type of enzymes do not obey Michaelis-Menten kinetics?
Allosteric Enzymes
What type of binding of enzyme-substrate results in a sigmoidal plot?
In allosteric enzymes, the binding of one substrate to an active site can alter the properties of the other active sites on the molecule -- facilitates cooperative binding, which is a sigmoidal plot.
What are the key regulators of metaboic pathways?
Allosteric enzymes
How does an irreversible enzyme inhibitor work?
It dissociates very slowly from its target enzyme because it has become very tightly bound to the enzyme
How does Penicillin work?
It is an irreversible enzyme inhibitor, working by covalently modifying the enzyme transpeptidase - preventing the synthesis of bacterial cell walls.
How does Aspirin work?
It is an irreversible enzyme inhibitor which covalently modifys the enzyme cyclooxygenase - reducing syntehsis of molecules involved in inflammation
What is reversible enzyme inhibition?
There is a rapid dissociation of the enzyme-inhibitor complex.
What is competitive inhibition?
The inhibitor binds to the same active site as the substrate
How does a competitive inhibitor work?
It diministhes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate
What is uncompetitive inhibition?
The inhibitor binds only to the substrate-enzyme complex (doesn't bind when substrate isn't present). The binding of substrate-enzyme creates the active site for the inhibitor. Uncompetitive inhibition cannot be overcome by adding more substrate
What types of inhibition can be overcome by adding more substrate?
Competitive inhibition (more substrate - more likely it will bind to enzyme rather than inhibitor binding to enzyme). Uncompetitive inhibition cannot be overcome by adding more substrate.
What is non-competitive inhibition?
The inhibitor and substrate have different active sites. Cannot be overcome by adding more substrate.
How can we determine whether a reversible inhibitor acts competitively or noncompetitively?
By using lineweaver-burke plot w/inhibitor, and comparing to plot w/out inhibitor.

competitive = same y-intercept (because Kmax doesn't change); greater x-intercept (b/c Km changes)

non-competitive = y-intercept is greater (because Vmax changes); same x-intercept

uncompetitive = parallel line shifted to the left (both Vmax and Km decrease)
What is k₁?
k₁ is the rate constant for enzyme-substrate complex formation; going from an enzyme and a substrate (E+S) to a complex (ES)
What is k-₁?
k-₁ is the reverse rate constant: going from enzyme-substrate complex (ES) to enzyme and substrate (E+S).
What is k₂?
k₂ is the rate constant for going from ES complex to enzyme and products (E+P)
What is k-₂?
The reverse rate constant: going from enzyme and product to ES complex. We usually ignore this (assume V₀), by assuming that products get used up once they're made, and therefore the rxn can't go in reverse. This is untrue, but makes the math easier.
In competitive inhibition, is there a change in Km of the enzyme for the substrate?
Yes: competitive inhibition changes Km but not Vmax
Can Vmax be attained in the presence of a competitive inhibitor?
Yes, if you add an excess of substrate
Which has tigher binding, and enzyme with a high or a low Km?
low Km
Is a competitive inhibitor able to affect binding of an enzyme with a high or low Km better?
A high Km, since it has weaker enzyme-substrate binding.
How do energy of activation of catalyzed (w/enzyme) and uncatalzyed reactions differ?
Catalyzed reactions have a lower activation energy, since the enzyme stabilizes the transition state.
What must we assume for Michaelis-Menten kinetics?
1. Steady State assumption:
ΔES/ΔT=O
So even if [E] and [P] are Δing
What does a second order reaction look like?
A + B →(k)→ P
V=k[A][B]
((units - m-'s-')
What does the formation of ES complex depend on?
Both the [S] and [E]:
more like a 2nd order reaction:
E+S ⇋ ES ⇋ E+P
What is the rate constant for the formation of ES?
k₁
What is the rate constant for the dissociation of ES to form E+P?
k₂
What is the rate constant for the dissociation of ES to form E + S?
k-₁
When must we assume that rate of ES formation and breakdown are equal?
Almost always: whenever we use M-M kinetics
k₁[E][S] = (k-₁ + k₂)/k₁ = km
What is the Michaelis constant?
km = k₁[E][S] = (k-₁ + k₂)/k₁
= shortcut to writing that we assume the breakdown and formation of ES complex are equal
What are the units of the Michaelis Constant?
Moles (concentration)
What can't we measure, which is why we use M-M kinetics?
[ES]. Therfore all equations must be rearranged to allow us to determine this concentration. We know how much enzyme and product we put into system, and we can usually see (color) or separate product.
How can we define [ES] in terms we can measure?
[Efree]=[Et]-[ES]
* We can plug in another equation for [ES]:
[ES]=([Et]-[ES])[S]/km
What is Vmax?
Vmax = k₂[Et] = all of enzyme is part of ES complex
What must we assume when doing Michaelis-Menten kinetics?
1. Steady-state assumption
2. product doesn't go back and alter reaction mechanism (we took away k-₂)
What is the equation of the Lineweaver-Burke Plot?
* Linearize by taking inverse of M-M:

1/V= Km/Vmax*S + 1/Vmax
What is the drawback of Lineweaver-Burke Plot?
Since most of the points are around the Y-intercept (1/Vmax), points that lie on the extreme (where [S] is low) can drastically alter the line (pulling up or down the slope)
Where do most of the points cluster in Lineweaver-Burke Plot?
Around the Y-intercept, which is the inverse of Vmax
What is the slope of Lineweaver-Burke Plot?
slope = Km/Vmax
What is the concentration of the substrate for a point that lies far to the right on a Lineweaver-Burke plot?
Low concentration of Substrate
What does Vmax reflect?
The turnover #: max. rate of catalysis
What is Kcat?
Kcat = k₂ @ Vmax: the fastest the enzyme can go
What is Km?
Reflects the affinity of E for S, but is not an affinity constant.
Low Km = tight binding
High Km = loose binding
What is Kcat/Km?
A measure of catalytic efficiency. It is the affinity for S and ability to catalyze a reaction.
units = m-'s-'
* limited by how fast an enzyme can get to the substrate: DIFFUSION
Why is Vmax lowered and Km remains the same in noncompetitive inhibition?
The inhibitor effectively lowers the concentration of active enzyme. The resulting solution behaves as if there was a more dilute solution of enzyme.
What is Ki?
The dissociation constant for a competitive inhibitor
What is the equation for Ki?
Ki=[E][I]/[EI]
What does a small Ki mean?
Small Ki = more potent inhibition (because most of the inhibitor is in the bound state rather than free)
What does a large Ki mean?
Large Ki = less potent inhibition, because more of the enzyme and inhibitor are free, rather than complexed
How do competitive inhibitors affect the Km value?
Adding a competitive inhibitor increases the Km, meaning more substrate is needed to get the same rxn rate.
How to uncompetitive inhibitors work?
They bind to the ES complex (ie only bind to enzyme when substrate is bound), forming an enzyme-substrate-inhibitor complex (ESI) - which does not go on to form any product.
What do uncompetitive inhibitors do to Km?
Uncompetitive inhibitors lower Km: inhibitor binds to ES to form ESI -- depleates ES. More S binds to E to maintain equilibrium of between E⇋ES, therefore lower [S] is required to form half the max [ES]: this causes a reduction in Km value.
What is noncompetitive inhibition?
Inhibitor binds to non-active site. Substrate can still bind, but no product can form from ESI.
What type of inhibition prevents substrate from binding to enzyme?
Competitive inhibition
What type of inhibition occurs when inhibitor binds to enzyme only when substrate is bound, preventing product formation?
Uncompetitive inhibition
What type of inhibition occurs when inhibitor binds to site other than the active site: substrate can still bind but no product is formed?
Noncompetitive inhibition
What is the kinetic effect of a noncompetitive inhibitor?
1. Km is unaltered.
2. Vm is decreased proportional to the amount of inhibitor
What is the kinetic effect of an uncompetitive inhibitor?
1. Km decreases
2. Vmax decreases
What is the kinetic effect of a competitive inhibitor?
1.Km increases
2. Vmax is unchanged
Why does noncompetitive inhibition lower Km?
[E] must be in equilibrium with [ES]. I binds to ES to form ESI, depleating ES. Therefore, a lower [S] is needed to form half of the max [ES]. -- reduces Km
What kind of inhibition cannot be overcome by adding more substrate?
Noncompetitive inhibition
What is a double-reciprocal plot useful for?
Distinguishing between competitive, uncompetitive, and noncompetitive
What is the plot of competitive inhibition?
1/V₀ versus 1/[S]
If Kcat decreases, what happens to Kcat/Km (a reflection of binding affinity)?
Decreases (looser binding)
If Km decreses, what happens to Kcat/Km (a reflection of binding affinity)
Increases (tighter binding)
What is the relationship between Km and affinity?
Inverse
What are the different types of inhibition?
Reversible:
* Competitive
* Uncompetitive
* Non-competitive

Irreversible: covalent bonding
What happens to the Vmax and Km in competitive inhibition?
Vmax = stays the same

Km (apparent) = increases: you need increased [S] to compete with I to get the same abount of binding
What happens to the Vmax and Km in uncompetitive inhibition?
Vmax = goes down (because you can't make as much product as fast)

Km = also goes down
What happens to the Vmax and Km in non-competitive inhibition?
Vmax = does not change (enzyme has same affinity for substrate whether or not inhibitor is bound, inhibitor only affects the ES -> E+P

Km = decreases
What does it mean if:
ΔG = 0
Equilibrium
What does it mean if:
ΔG < 0
Exergonic: reaction will occur spontaneously
What does it mean if:
ΔG > 0
Endergonic: needs an input of energy for reaction to occur
Does ΔG correlate with rate of reaction?
NO
What is a double-displacement (ping-pong) reaction?
Substrate 1 in - Product 1 out - Substrate 2 in - Product 2 out
What type of inhibition is occuring if
Vmax-stays the same
Km-increases
Competitive Inhibition
What type of inhibition is occuring if
Vmax-decreases
Km-decreases
Uncompetitive Inhibition
What type of inhibition is occuring if
Vmax-decreases
Km-stays the same
Non-Competitive
What happens to Km in
1. competitive
2. uncompetitive
3. non-competitive
1.comeptitive - Km increases
2.uncompetitive - Km decreases
3.non-competitive - stays same
What happens to Vmax in
1. competitive
2. uncompetitive
3. non-competitive
1.competitive - Vmax stays same
2.uncompetitive - Vmax decreases
3.non-competitive - Vmax decreases
What is the suffix added to denote something is an enzyme?
-ase
What are the six major classes of enzymes?
1. oxidoreductases
2. transferases
3. hydrolases
4. lyases
5. isomerases
6. ligases
What do oxidoreductases do?
oxidaton and reduction
What do transferases do?
group transfer
What do hydrolases do?
Hydrolysis reactions (transfer of functional groups to water)
What do lyases do?
Addition or removal of groups to form double bonds
What do isomerases do?
Isomerization = intramolecular group transfer
What do ligases do?
Ligation of two substrates at the expense of ATP hydrolysis
What does a graph of competitive inhibition look like?
Competitive Inhibition
What does a graph of non-competitive inhibition look like?
Non-competitive Inhibition
What does a graph of un-competitive inhibition look like?
Uncompetitive inhibition
What does Km tell us?
The Km is the substrate concentration yielding a velocity of 1/2Vmax
How can we determine what functional groups are required for enzyme activity?
X-ray crystallography of the enzyme bound to its substrate. Irreversible inhibitors modify these functional groups (covalent linkage).
What are the three categories of irreversible inhibitors?
1. group-specific reagents
2. reactive substrate analogs (affinity labels)
3. suicide inhibitors
What are group-specific reagents?
One type of irreversible enzyme inhibitor.
- React with specific side chains of amino acids.
What are two examples of group-speific reagents (a type of irreversible inhibitor that reacts withs specific side chains of amino acids)
1. DIPF - diisopropylphosphofluoridate: modifies only one of 28 ser residues in chymotrypsin.

2. iodoacetamide
What are affinity labels (reactive substrate analogs)?
A type of irreversible enzyme inhibitors. Structurally similar to the substrate of the enzyme - covalently binds to active-site residue.
How does iodoacetamide (an irreversible, group-specific reagent) react with an enzyme to inhibit it?
Enzyme + Iodoacetamide → Inactivated enzyme
Competitive Inhibition
What type of inhibition is this?
Uncomptetitive Inhibition
What type of inhibition is this?
What does enzyme inhibition using DIPF look like? What type of inhibition is this?
DIPF is a group-specific irreversible inhibitor. It works by covalently modifying a crucial serine residue.
What is a suicide inhibitor (mechanism-based inhibitor)?
Suicide inhibitors are irreversible inhibitors that are the most specific way to inhibit an enzyme. They are a modified version of the substrate that initially binds to the active site of an enzyme and processed normally. The enzyme-substrate reaction however, generates a chemically reactive intermediate taht inactivates the enzyme through covalent modification.
What is a modified substrate called, which, when it binds, catalyzes the formation of a reactive intermediate that covalently modifies an enzyme?
A suicide inhibitor.
What is an example of a suicide inhibitor?
Some MAOIs, such as (-)deprenyl, which is used to treat Parkinsons disease and depression.
What is (-)deprenylWhat does (-)deprenyl look like?
(-)deprenyl is an MAOI. It inhibits monoamine oxidase by preventing the binding of FAD, a necessary cofactor. This is done by attacking the covalently modifying the flavin prosthetic group after the inhibitor has been oxidized.
Who proposed that compounds resemblind the transition state of catalyzed reaction should be effective inhibitors of enzymes?
Linus Pauling
What did Linus Pauling propose?
Compounds resemblind the transition state of catalyzed reaction should be effective inhibitors of enzymes
What is a transition-state analog?
A compound that resembles the transition state of a catalyzed reaction. Serve as effective enzyme inhibitors
What is the mechanism of inhibition by a transition-state analog
Inhibition by transition-state analog. Example: L-Proline to D-Proline
What is a good thing to synthesis if you want a potent, irreversible enzyme inhibitor?
Rather than synthesizing something that looks like the substrate, making something that looks like the transition state of the substrate works better, because there is extremely selective binding of the transition state.
What is an abzyme?
A catalytic antibody
How are abzymes produced?
Using transition-state analogs as antigens.
What do studies with transition-state analogs tell us?
That enzymes can function by assuming a conformation in the active site that is complementary in structure to the transition state
What 3 thigns can transition state analogs do?
1. sources of insight into catalytic mechanisms

2. serve as potent and specific enzyme inhibitors

3. can be used as antigens to generate a wide range of novel catalysts
What was the first antibiotic discovered?
Penicillin
How does Penicillin inhibit bacterial growth?
It inhibits the cross-linking transpeptidase, which prevents cross-linking of different petidoglycan strands of the cell wall of bacterium.
- irreversible inhibition: cell-wall synthesis cannot take place.
How does penicillin inhibit transpeptidase?
Irreversible - suicide inhibitor:
1. binds to transpeptidase
2. serine residue at active site of transpeptidase attacks carbonyl on penicillin
3. this makes penicilloyl-serine derivative which can't continue in cell-wall synthesis
What tells us about enzyme efficiency?
Kcat/Km
How does varying the concentration of an enzyme effect its Lineweaver-Burke plot?
Same x-intercept, different y-intercept, since Vmax increases with increased concentration of enzyme.
Which is the rate limiting step in conversion of A to D, one with higher or lower Km?
The one with the higher Km would be the rate limiting step, since the enzyme has looser binding to the substrate, so it can't catalyze a reaction as fast compared to one with tighter binding.
What does non-competitive inhibition look like?
Non-Competitive Inhibition
What does the reaction showing DIPF inhibiting Acetylcholine esterase look like?
Diisopropyl phosphofluoridate (DIPF) is an inhibitor of acetylcholine esterase
What does V0 equal when [S] is very large?
Enzyme is saturated with substrate:

V0= Vmax*[S] / Km + [S]
What does V0 equal when [S] is very small?
When [S] is very small, Km + [S] approximately equals Km, so:

V0= Vmax*[S] / Km
What does V0 equal when [S]=Km?
V0= Vmax*[S] / [S] + [S]


= Vmax/2
What is Vmax a measure of?
Vmax is a measure of how fast the enzyme can go at full speed
In a first order reaction, what is the rate dependent on?
Substrate concentration (and K, the rate constant)
Is metal a component of the active site in NMP kinase?
No
Is metal a component of the active site in restriction endonucleases?
Yes
Is metal a component of the active site in carbonic anhydrase?
Yes
Is metal a component of the active site in metalloproteases?
Yes
How does the binding of Magnesium ion to the nucleotide (ex: ATP) enhance catalysis?
The interaction between the magnesium ion and the phosphoryl group oxygen atoms hold the nucleotide in a well-defined conformation that can be bound by an enzyme in a specific way.
What does metal ion do in NMP catalyis?
It binds to the oxygen of the phosphoryl group of the substrate (ex: ATP) - this holds the substrate complex so that the enzyme can bind properly. CONFORMATION!
How does the binding of Magnesium ion to the nucleotide (ex: ATP) enhance catalysis?
The interaction between the magnesium ion and the phosphoryl group oxygen atoms hold the nucleotide in a well-defined conformation that can be bound by an enzyme in a specific way.
What does metal ion do in NMP catalyis?
It binds to the oxygen of the phosphoryl group of the substrate (ex: ATP) - this holds the substrate complex so that the enzyme can bind properly. CONFORMATION!