Ch 6: The Behavior of Proteins: Enzymes

Thermodynamic Principles of Enzymes

I. Know the general characteristics of enzymes as biological catalysts
a. Enzymes are made up of globular proteins and in some cases, ribozymes.
b. Influence on reaction rate. By up to 1020.
c. Highly specific – can distinguish stereoisomers of a compound
d. Regulated processes
II. Know the standard state conditions
a. Standard free energy (∆𝐺) – the difference between the energy of the reactants and products under standard
conditions.
i. Enzymes DO NOT alter equilibrium: reaction rate and thermodynamic favorability are two different topics
1. Enzymes can speed up reactions but cannot alter the equilibrium constant or free energy change.
a. Rate of a reaction is a kinetic property; ΔG is a thermodynamic property: unrelated

ii. The reaction rate depends on the activation energy (ΔG°‡) – energy required to start a reaction
1. The activation energy of an uncatalyzed reaction requires more
energy to start than a catalyzed reaction
III. Understand the concept of activation energy and the transition state
a. The activation energy is the energy needed to reach transition state
i. “Spontaneous” reactions are not instantaneous – they need to overcome
an energy barrier, too.
ii. Transition state lies in the maximum point of the curve, connecting the
reactants and products
1. TS contains the necessary amount of energy and correct atom
order to produce product
b. The activation energy profile shows the intermediate stages of a reaction, those
between initial reactants and final products/states at which old bonds break and
new bonds form.
i. The activation energy directly affects the reaction rate and the presence of a catalyst speeds up a reaction
by changing the mechanism, thus lowering the activation energy
1. Enzymes lower the activation energy needed for a substrate to reach TS, but do not change G
IV. Know the relationship between temperature and catalysis
a. Rate increases with temperature – raising the temperature increases the energy available for reactants to reach TS
i. BUT, increase of a reaction rate only occurs for a limited extent.
1. Raising temp too much causes heat to denatures enzyme, which slows down the reaction.
a. Thermal energy > bond energy.

Review Exercises Recommended: 2, 4, 5, 6, 7, 10, 11, 12

1. 2) Most enzymes are proteins but some catalytic RNAs, such as ribozymes are known.
2. 4) Give two reasons why enzyme catalysts are103 to 105 more effective than reactions that are catalyzed by, for example,
simple H+ or OH- .
a. Enzymes hold substrates in favorable spatial positions – in close proximity
b. Enzymes can bind effectively to the transition state to stabilize it
i. Note: ALL catalysts lower activation energy so this is not a particular enzyme function
3. 5) For the reaction of glucose with oxygen to produce carbon dioxide and water, the change in free energy is -2880 kJ mol-1,
a strongly exergonic reaction. However, a sample of glucose can be maintained indefinitely in an oxygen-containing
atmosphere. Reconcile these two statements.
a. Because of the negative change in free energy, the reaction of glucose w/ oxygen is thermodynamically favored. The
fact that glucose can be maintained in an oxygen atmosphere is a reflection of the kinetics in the reaction, requiring
overcoming an activation barrier.
4. 6) Would nature rely on the same enzyme to catalyze a reaction either way (forward or backward) if the change in free
energy were -0.8 kcal per mol ? If it were -5.3 kcal per mol?
a. First one: Yes; local concentrations (Le Chatieler’s principle) could easily dictate the direction
b. Second one: No. Local concentrations would seldom be sufficient to overcome the relatively large change in free
energy of 5.3 kcal in the reverse direction.
5. 7) Suggest a reason why heating a solution containing an enzyme markedly decreases its activity. Why is the decrease of
activity frequently much less when the solution contains high concentrations of the substrate?
a. Heating a protein denatures it. Enzymatic activity depends on the correct 3D conformation of the protein
i. The presence of bound substrate can make the protein harder to denature.
6. 10) What effect does a catalyst have on activation energy?
a. Lowers activation energy
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 Ch 6: The Behavior of Proteins: Enzymes
7. 11) An enzyme catalyzes the formation of ATP from ADP and phosphate ion. What is its effect on the rate of hydrolysis of
ATP to ADP and phosphate ion?
a. Enzymes, like all catalysts, increase the rate of the forward and reverse reaction to the same extent.
8. 12) Catalysts do NOT increase the amount of product nor do they alter change in free energy.

Enzyme Reaction Rates

I. Know how to express enzyme reaction rate as loss of substrate or increase in product concentration over time
a. Rate of disappearance of one of reactants:
Rate of disappearance of A =  Δ  A  /Δt
Rate of disappearance of B =  Δ  B /Δt

b. Rate of appearance of product

Rate of appearance of P = Δ  P  /Δt

II. Understand the concept of the rate or proportionality constant

a. The negative signs for the change in [A] and [B] indicate that A and B are being used up, while [P] is produced.
b. The rate of a reaction at a given time is proportional to the product of the concentrations of the reactants raised to the
appropriate powers.
i. Rate = k[A]f[B]G
1. Rate constant = K
2. Exponents are equal to the number of molecules involved in the detailed steps
III. Know how to determine the order of a reaction
a. With respect to each substrate
b. Overall order of reaction: sum of all exponents
i. Rate = k[A]1[B]1 is first order w/ respect to A, first order with respect to B, and second order overall.
ii. Exponents in a reaction may equal zero
1. The rate of zero order depends not on concentrations of reactants but the presence of catalysts.
IV. Know what constitutes a zero order reaction – rate = k[A])= k
a. How enzymes achieve this state ([S] >>>[E]) – the [reactant] is much greater than [enzyme]
i. Enzyme catalyzed reactions exhibit zero order kinetics when the concentrations of reactants are so high that
the enzyme completely saturated the reactant molecules.
ii. Despite the amounts of reactants added, the reaction proceeds at the same rate.

Review Exercises Recommended: 13, 16

1. Suggest a reason for carrying out enzymatic reactions in buffer solutions

a. Enzymes have a fairly sharp pH optimum value. It’s necessary to ensure that the pH of the reaction mixture remains
at that optimum value. This is especially true for reactions that require or produce H ions.

Enzyme-Substrate Binding
I. Know the general principles of binding of substrates to enzyme
a. Enzymes bind at active site – situated in a cleft or crevice in the protein and consists of amino acids essential for
enzymatic activity.
b. Types of intermolecular forces involved –
i. Substrate binds to active site by noncovalent interactions between substrate, side group, and backbone
groups of amino acids making up the active site
c. The two models for binding
i. Lock and key – assumes a high degree of similarity between the shape of the substrate and the geometry of
the binding site on the enzyme.
1. The substrate binds to a site whose shape complements its own
a. Does NOT take into account an important protein’s property: conformational flexibility.
ii. Induced fit – takes into account that proteins have some 3D flexibility
1. Binding of a substrate induces a conformational change in the enzyme that results in a
complementary fit after the substrate is bound.
2. This model makes more sense in terms of the nature of TS and the lowered activation energy that
occurs with enzyme catalyzed reactions.
3. Mimics the transition state.
d. Energy changes associated with formation of ES complex
i. Enzyme + substrate must bind to form the ES complex before the reaction can occur

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 Ch 6: The Behavior of Proteins: Enzymes
1. An attraction MUST exist b/w E and S for them to bind
a. This attraction causes the ES complex to be lower on an energy diagram than E + S at the
start.
b. The bound ES must attain the conformation of TS enzyme transition state complex (EX).
ii. If E and S fit perfectly like the lock and key model states, then –
1. When the enzyme and the substrate combine to form a complex, the attraction bringing them
together would put the enzyme-substrate complex at a lower energy than the starting individual
components of the enzyme and the substrate.
a. Essentially, the rate of the
reaction would decrease because
the enzyme-substrate complex
would form a lower energy,
increasing the activation energy
required to reach the transition
state.
b. The lock and key model
increases the distance to the
transition state instead of
minimizing it, thereby rendering an unstable reactant which does not undergo catalysis.
iii. Enzymes increase rate of reaction by lowering the energy of the transition state, EX, while raising the
energy of the ES complex, supporting the induced fit model.
e. Importance of proximity and orientation to speed of reaction
i. 1) Substrate binds; 2) transition state is formed; 3) catalysis can occur
1. In TS, the substrate is placed in the correct orientation w/ respect to the atoms it needs to bind to in
order to react.
a. Proximity and orientation speed up the reaction.
2. Once product is formed, substrate is released from the enzyme, which can move onto more.
II. Know the changes in structure and energy associated with product formation and release

Review Exercises Recommended: 19, 20, 21

1. 17) Distinguish between the lock and key model and induced fit model for binding of a substrate to an enzyme
a. Lock and key – substrate fits into a comparatively rigid protein that has an active site w/ a well-defined shape
b. Induced fit – enzyme undergoes conformational change on binding to the substrate
i. The active site takes shape around the substrate.
2. 19) Other things being equal, what is a potential disadvantage of an enzyme having a very high affinity for its substrate?
a. The ES complex would be in an “energy trough,” with a large activation energy to the transition state.
3. 20) Amino acids that are far apart in the amino acid sequence of an enzyme can be essential for its catalytic activity. What
does this suggest about its active site?
a. Amino acids far apart in the amino acid sequence can be brought closer together in the tertiary 3D structure of a
protein due to protein folding.
i. The critical amino acids are in the active site.
4. 21) If only a few of the amino acid residues of an enzyme are involved in its catalytic activity, why does the enzyme need
such a large number of amino acids?
a. The overall protein structure is needed to ensure the correct arrangement of amino acids in the active site

Steady State Kinetics – condition in which the [enzyme-substrate complex] remains constant in spite of continuous turnover.
I. Steady state is reached when the rate of formation of the enzyme-substrate complex = rate of its breakdown.
a. The rate of the reactions that lead to and away from [ES] formation is equal.
II. Know the simple mechanism for a one-substrate/one-product reaction
a. Know the three rate constants and to what they relate
k
E + S ⇀! !!k 1!!⇀! ES ¾k¾
2
® E+P
i. -1

1. K1 – rate constant for the formation of ES complex

2. K-1 – rate constant for the dissociation of ES complex to free E + S; reversible
3. K2 – rate constant for conversion of ES complex  Product (E+P)

b.Know the basic assumptions in this model:

i. One substrate and one product involved
ii. Only one catalytic step required to convert substrate to product that is irreversible
III. Know the relationship between velocity and substrate concentration
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 Ch 6: The Behavior of Proteins: Enzymes
a. Hyperbolic plot – levels off at Vmax
b. Initial velocity is first order (why?) – velocity/rate depends on substrate concentration
i. All calculations assume that the velocity/rate measured is the initial velocity.
ii. At low [substrate] levels, the reaction is 1st order, which implies that velocity/rate depends on the substrate
concentration
1. In linear portion, activity relates to [substrate] – indicates 1st
order b/c it depends on the concentration of one reactant.
c. Reaches Vmax and zero order at high [S] – Vmax is reached thus velocity/rate is
independent of substrate concentration
i. At higher substrate levels, where the enzyme is saturated, the constant
reaction rate is zero order.
ii. The flat area of the curve indicates that as the [substrate] increases, the
rate does not change.
1. Therefore, rate is independent of concentration. The active sites
of all the enzyme molecules are fully saturated with substrate.
a. This constant rate in which the enzyme is saturated w/
substrate is the Vmax
i. When the reaction is ½ its max value, [substrate] is equal to Michaelis constant.
IV. Know the significance of KM
a. Equilibrium constant that measures substrate affinity:
i. Low Km = higher binding affinity for the substrate (association w/ substrate is favored).
1. It takes less of the substrate to be present for the enzyme to bind
a. Reaction proceeds with [low substrate] if the enzyme has a high affinity for substrate.
ii. The substrate concentration at which the reaction proceeds at ½ of its maximum velocity = K M
1. At KM, the speed of product formation is at ½ its maximum value (V maz)
V. Be able to write expressions for rate of formation and breakdown of ES
∆[𝐸𝑆]
a. Rate of formation = = 𝑘1 [𝐸][𝑆]
∆𝑡
i. Change in the concentration of ES overtime
−∆[𝐸𝑆]
b. Rate of breakdown = = 𝑘−1 [𝐸𝑆] + 𝑘2 [𝐸𝑆]
∆𝑡
i. Negative denotes decreasing ES concentration overtime
1. Substrate dissociates from enzyme before forming product
2. Substrate gets converted to product

VI. Know the definition of steady state kinetics

a. Steady state: condition in which there is no change in the concentration of ES in spite of continuous turnover
i. The enzyme remains saturated with substrate and spends no appreciable time in the free or unbound form
1. Thus, the rate of formation of ES = rate of breakdown of ES
ii. Can only occur if the [Substrate] >> [Enzyme]T
1. No change in [ES] over time
VII. Know the relationship between enzyme concentration and velocity
a. More enzymes  higher reaction velocity

Review Exercises Recommended: 22, 23

1. Show graphically how the reaction velocity depends on the enzyme concentration. Can a reaction be saturated with enzyme?
a. The reaction velocity remains the same with increasing enzyme concentration. Its theoretically possible but highly
unlikely that the reaction will be saturate with the enzyme.
2. Define steady state, and comment on the relevance of this concept to theories of enzyme reactivity.
a. Steady state – assumes that the concentration of the enzyme-substrate complex does NOT change appreciably over
time in which the experiment takes place
i. The rate of appearance of the complex is set to equal its rate of disappearance, simplifying the equation for
enzyme kinetics.

Michaelis-Menten Equation
I. Michaelis-Menten Model – confirms that catalysts (like enzymes) are regenerated and re-useable.
a. Know that Michaelis constant, KM, is a collection of rate constants
i. Km is an equilibrium dissociation constant.

b. Know that when [S] >>> [E], [ES] = [E]t and Vmax = k2[E]t
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 Ch 6: The Behavior of Proteins: Enzymes
i. In steady state conditions, the [substrate] is much greater than [enzyme]; all enzyme molecules are bound to
substrate.
1. This means that [ES] = [enzyme total]
a. These conditions are under zero order conditions and Vmax is reached.
b. Initial velocity = Vmax
𝑉 [𝑆]
2. Michaelis-Menton equation: 𝑉0 = 𝑚𝑎𝑥
𝐾𝑀 +[𝑆]
a. V) is directly dependent on Vmax – how fast the enzyme converts substrate to product
b. V) is inversely related to Km – as Km ↓ and substrate affinity ↑, more product is made per
unit time.
c. All calculations assume that the velocity/rate measured is the initial velocity.
d. At low [substrate] levels, the reaction is 1st order, which implies that velocity/rate depends on the substrate
concentration
e. At higher substrate levels, where the enzyme is saturated, the constant reaction rate is zero order.
i. Therefore, rate is independent of concentration. The active sites of all the enzyme molecules are saturated.
ii. This constant rate in which the enzyme is saturated w/ substrate is the Vmax
1. When the reaction is ½ its max value, [substrate] is equal to Michaelis constant.
iii. At infinite [substrate] the reaction would proceed at its maximum velocity, Vmax.
II. Know the significance of the Michaelis-Menten equation and how to use it
a. You do NOT have to know how to derive it
b. Know that when initial velocity, V0 = ½ Vmax, [Substrate] = KM
i. When [substrate] = KM, initial velocity is equal to half of the maximum
velocity, which is equal to Km.
ii. KM is an inverse measure of the affinity of the enzyme for the substrate:
1. The lower the KM, the higher the affinity
c. Know how to estimate Vmax and KM from a substrate saturation curve
i. The curve that describes the rate of a non-allosteric enzymatic reaction is
hyperbolic
1. B/c Vmax is an asymptote, the equation is transformed to a linear
form by reciprocal-ing it.
III. Michaelis constant – the strength of binding of a substrate to an enzyme.
a. When the rate of a reaction is ½ its maximum value, the substrate concentration is equal to Michaelis constant, Km
IV. How does michaelis equation relate maximum enzyme speed and the affinity for substrate?

Review Exercises Recommended: 25

1. For an enzyme that displays Michaelis–Menten kinetics, what is the reaction velocity, V (as a percentage of Vmax), observed
at the following values?
𝑉𝑚𝑎𝑥[𝑆]
𝑉=
𝐾𝑚 + [𝑆]
a. When [S] = Km, V = ½ Vmax When [S] = ½ Km, V= 1/3 Vmax When [S] = 2Km, V = 0.67 Vmax
𝑉𝑚𝑎𝑥[𝑆] 1
b. When [S] = 0.1 Km, V = 0.09Vmax  𝑣 = 𝑆 = 𝑉𝑚𝑎𝑥 × = 0.91; 1 − 0.91 = 0.09.
[ ]+[𝑠] 1.1
10
c. When [S] = 2Km, V = 0.67 Vmax When [S] = 2Km, V = 0.67 Vmax
𝑉𝑚𝑎𝑥[𝑆] 1
d. When [S] = 10 Km, V= 0.91 Vmax 𝑣 = 𝑆 = 𝑉𝑚𝑎𝑥 × = 0.91
[ ]+[𝑠] 1.1
10
Enzyme Mechanisms and Lineweaver-Burk
I. Know the definition and types of Bi-Bi reactions; occur with two substrates and give two products
a. Ordered mechanism = substrates have to bind the enzyme in a specific
order
b. Random = substrates can bind to the enzyme in any order
c. Ping-pong = a substrate binds the enzyme and releases a product before
the second substrate can bind.
i. The enzyme is covalently modified in the process and must be
undone before moving onto next substrate
1. This type of enzyme is used when transferring a
functional group from one molecule to another
II. Know the significance of the Lineweaver-Burk equation and be able to use it
a. Know what an L-B plot looks like and how to determine Vmax and KM
from the graph
i. Line-weaver Burke Double-reciprocal plot of enzyme kinetics

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 Ch 6: The Behavior of Proteins: Enzymes
1 𝐾𝑚 1 1
= ( )+ in the form of y=mx+b
𝑉 𝑉𝑚𝑎𝑥 𝑆 𝑉𝑚𝑎𝑥
1. Make sure to use the reciprocal of substrate concentration and velocity
b. Know the kinetic values associated with the slope and x- and y-intercepts
c. Km – substrate concentration where velocity is at half its max value
I. Review Exercises Recommended: 26, 27*, 33, 39, 40, 41
II. 26) Determine the values of Km and Vmax for the decarboxylation of a -keto acid given the following data.
Substrate Concentration Velocity

2.500 0.588

1.000 0.500

0.714 0.417

0.526 0.370

0.250 0.256
a. Vmax = 0.681 mM min-1
1
i. Vmax =
𝑌−𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡
Km = 0.421 M
Km = slope x Vmax

I. 27) The kinetic data in the following table were obtained for the reaction of carbon dioxide and water to produce
bicarbonate and hydrogen ion catalyzed by carbonic anhydrase: CO2+H2O  HCO3--+H+

From these data, determine Vmax and Km for the reaction.

CO2 Concentration 1/Velocity ( sec)

1.25 36 X 10^3

2.5 20 X 10^3

5.0 12 X 10^3

20.0 6 X 10^3

*The textbook solution to this problem gives you the correct value for V max but not for KM. The latter should be 0.010 M.
a. Vmax = 2.5x10-4
b. Km = 0.01 M
II. 33) Plotting a linear line > curve for enzymatic reactions because its easier to detect variations in individual points
III. 39) What are the three most common mechanisms for enzyme-catalyzed reactions that have two substrates?
a. Ordered, ping pong, random
IV. 40) Ping pong VS ordered
a. Ping pong -
b. Ordered -
V. 41) How do scientists determine the of a substrate that is part of an ordered reaction with two substrates?
a. The trick to determining the Km of one of them is to run the reaction w/ saturating concentrations of the other one

Kinetic Parameters
I. Know the significance of KM
a. Substrate concentration where reaction half-maximal
i. If the [S] is much less than Km, the enzyme is not working efficiently
b. Good estimate of [S] in vivo
c. Dissociation constant and inverse measure of substrate affinity
i. Km is the rate at which [ES] dissociates compared to the rate it associates:
1. Thus, it’s a true dissociation constant.
II. Know the significance of kcat and its relationship to Vmax
a. Catalytic rate constant (Kcat) is the turnover number – amount of product formed per enzyme per unit time

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 Ch 6: The Behavior of Proteins: Enzymes
𝑉𝑚𝑎𝑥
b. K2 = Kcat =
[𝐸𝑛𝑧𝑦𝑚𝑒𝑇𝑜𝑡𝑎𝑙]
i. Kcat is the maximum # of substrate molecules that an enzyme can convert into product at its max speed
1. Greater Kcat means that more product can be produced per unit time
a. Kcat correlates directly with Vmax; Vmax = (Kcat)(Enzyme total)
2. High K = low Km = high affinity  increase in an enzyme’s catalytic efficiency
III. Biological examples
a. Low Km and low Kcat: lysozyme
i. Kcat indicates slow conversion rate
ii. Km indicates a much higher affinity – finds bacterial invaders at very low concentrations
b. High Km and high Kcat: carbonic anhydrase
i. Kcat indicates fast conversion rate to maintain blood pH
ii. Km indicates a low affinity

Review Exercises Recommended: 24, 31

I. 31) You do an enzyme kinetic experiment and calculate a Vmax of 100µmol of product per minute. If each assay used
0.1 mL of an enzyme solution that had a concentration of 0.2 mg/mL, what would be the turnover number if the enzyme had
a molecular weight of g/mol?
a. 1.56x10-10 number of moles of enzyme
b. Turnover number = 10,700 sec-1

Examples of Enzyme-Catalyzed Reactions

I. Know the example of chymotrypsin
a. The reaction it catalyzes – hydrolyzes peptide bonds on carboxyl sides of aromatic amino acids, Phe, trp, tyr
i. Can also hydrolyze an ester bond R-O-R + H2O acid— + alcohol (R-OH)
b. P-nitrophenyl ester’s reaction rate depends on [S], which is [P-nitrophenyl ester]
i. Chymotrypsin breaks the ester bond releasing acetic acid and paranitrophenolate
1. Its resonance structure allows it to absorb light in the visible range to give varying intensities of
yellow, which measures product concentration
ii. Hyperbolic plot of Velocity vs. [S]
1. Enzyme obeys the michaelis menton model of kinetics
II. Know the example of Aspartate Transcarbomylase (ATCase)
a. The reaction it catalyzes: allosteric enzyme that catalyzes early reaction in pyrimidine biosynthesis
b. Carbomyl phosphate + aspartate  carbamoyl aspartate + HPO42-
i. Catalyzed by ATCase
c. Exhibits cooperativity
i. Sigmoidal plot of Velocity vs. [S]
ii. Follows allostery

Review Exercises Recommended: 44, 46

1. 44) Do all enzymes display kinetics that obey the Michaelis–Menten equation? Which ones do not
a. No. Kinetic behavior of allosteric enzymes do not obey it
2. 46) If we describe an enzyme like ATCase and say that it exhibits cooperativity, what do we mean?
a. Enzymes w/ cooperativity have multiple subunits that can influence each other
i. Most enzymes that are cooperative exhibit positive cooperativity which means that the binding of substrate
to one subunit will make it easier to bind the substrate to another subunit.

Competitive Inhibition
Know the definition of inhibitor and reversible inhibitor
a. Reversible inhibitors – bind temporarily and alter enzyme’s function only while bound
i. Affect enzyme activities in various ways
II. Know the characteristics of competitive inhibitors – a type of reversible inhibitor
a. To what form of the enzyme they bind and where
i. Compete for binding in the active site.
ii. The enzyme can in two mutually exclusive equations – there is competition for which it binds
1. Enzyme binds substrate: E + S ⇌ ES
2. Enzyme binds inhibitor: E + I ⇌ EI
b. Can be overcome by adding more substrate
c. Often resemble either substrate or transition state

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 Ch 6: The Behavior of Proteins: Enzymes
[𝐸][𝐼]
III. Know the significance of KI =
[𝐸𝐼]
a.Inverse measure of enzyme affinity for inhibitor
i. As the value of Ki decreases, the enzyme is MORE likely to bind the inhibitor
1. Thus, the inhibitor’s potency decreases.
IV. Know which kinetic parameters change and how (and why)
a. Know the significance of and how it relates to inhibition
b. The inhibitor competes for substrate binding
i. If substrate binds the inhibitor, the inhibitor does not impact the conversion to product
1. Thus, competitive inhibitor does NOT alter Vmax
c. Competitive inhibitor does influence substrate binding, lowering the effective affinity of the enzyme for substrate
i. Competitive inhibitor increases Km.
[𝐼]
1. Alpha = 1 +
𝐾𝐼
a.Alpha is directly related to [inhibitor] –
i. The more inhibitor added, the greater the affect will be
b. As Ki decreases, alpha increases
i. The higher affinity in which the enzyme binds the inhibitor, the more it will
perturb enzyme function
V. Know how to recognize a competitive inhibitor, graphically
a. Does not alter y-intercept; thus, Vmax is unchanged
b. Increases slope w/ more inhibitor added
c. X-intercept is decreasing because Km increases in the
presence of competitive inhibitors
i. Moves closer to zero or the point of origin.
d. Increasing [substrate] can only overcome competitive
inhibitors
VI. Competitive inhibitor : Lowers substrate affinity (Km) but has
no impact on Vmax
VII. Non-competitive inhibitor: no impact on Km but lowers Vmax

Noncompetitive Inhibition – reversible inhibitor

I. Know the characteristics of noncompetitive inhibitors
a. To what form of the enzyme they bind and where
i. Binds to the enzyme in a location outside of the active site; it distorts the active site to inhibit the reaction
1. Thus, it can bind whether the substrate is bound or not, causing a conformational change
a. Renders the active site to be less able to bind to substrate
b. Multiple equilibria occur
b. Cannot be overcome by adding more substrate: there is no competition
II. Know which kinetic parameters change and how (and why)
a. Does NOT alter Km value b/c it does not interfere with
substrate binding
b. Inhibitor does prevent product formation while bound even if
substrate is present
i. Vmax decreases because once its bound, it can
make less of the product
1. By how much? Changes by the factor of
alpha
ii. Once its released, product formation rate increases
III. Know how to recognize a noncompetitive inhibitor, graphically
a. Slope and Y-intercept increase because Vmax is lowered
b. X-intercept is unchanged b/c Km is unchanged
IV. Gives the impression that there is less enzyme present.

Mixed Noncompetitive Inhibition

I. Know the characteristics of mixed noncompetitive inhibitors
a. To what form of the enzyme they bind and where
i. Can bind free enzyme [E] or ES complex – multiple equilibria occur
b. Cannot be overcome by adding more substrate
II. Know which kinetic parameters change and how (and why)

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 Ch 6: The Behavior of Proteins: Enzymes
a.Unlike a noncompetitive inhibitor, a mixed noncompetitive inhibitor DOES affect binding of substrate affinity, Km
i. It also lowers Vmax
III. Know how to recognize a mixed noncompetitive inhibitor, graphically
a. Increase slope and Y-intercept because it lowers Vmax
b. X-intercept changes because Km changes, moving it closer to
the origin.
IV. Know how they differ from purely noncompetitive inhibitors
a. Mixed noncompetitive inhibitor may increase or decrease Km
but it always decreases Vmax

Uncompetitive and Irreversible Inhibition

 Know the characteristics of uncompetitive inhibitors – reversible
o To what form of the enzyme they bind and where –
 This reversible inhibitor ONLY can bind to ES
complex (center), never the free enzyme
 Since it binds to the enzyme in the presence of
a substrate, it blocks the catalytic step,
decreasing Vmax
o Cannot be overcome by adding more substrate
 Know which kinetic parameters change and how (and why)
o Vmax decreases causing a lower rate of product formation
o Uncompetitive inhibitor does NOT influence substrate binding
 Instead, it LOWERS the value of Km, increasing substrate binding affinity – helpful.
 This occurs b/c of Le Châtlier’s principle – system acts to relieve stress
 The amount of ES is lowered b/c the inhibitor is converting it to the EIS complex
 The system compensates by generating more ES from E+S
o It ultimately lowers enzyme activity
 Know how to recognize an uncompetitive inhibitor, graphically
o B/c Vmax decreases, slope and y-intercept increase by the
alpha factor
 But it decreases Km by the same alpha factor
amount
o Produces parallel lines with increasing slopes – line is
parallel and shifted down closer to origin
 No intersections.
 Understand the general concept and types of irreversible inhibitors
o Covalent binding – permanent inactivation lowers the
effective [[enzyme]
o Suicide substrates – molecules that combine at enzyme
active site that cannot be released
 Bind irreversibly and inactivate it – stuck in place
 Trojan horse substrate – sneak into active
site and damage it
 Used in medicine such as penicillin

Review Exercises Recommended: 47, 48, 49, 53, 54, 57, 58, 59, 60, 61, 62

I. How can competitive and pure noncompetitive inhibition be distinguished in terms of ?

a. Competitive inhibition – Km increases
b. Noncompetitive inhibition – Km doesn’t change
II. Why does a competitive inhibitor not change Vmax?
a. Competitive inhibitors block binding, not catalysis.
III. Why does a pure noncompetitive inhibitor not change Km?
a. Because it does not change the affinity of an enzyme for its substrate.
IV. Where do lines intersect on a Lineweaver–Burk plot showing competitive inhibition?
a. Intersect at y-axis intercept which equals 1/Vmax
b. On a Lineweaver–Burk plot showing inhibition?
i. Intersect at x-axis intercept, which equals -1/Km
V. What is the difference between pure and mixed noncompetitive inhibition?

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 Ch 6: The Behavior of Proteins: Enzymes
a. Pure Noncompetitive inhibition – does not change the affinity of the enzyme for substrate at all
i. Km does NOT change
b. Mixed – substrate and inhibitor affect one another such that Km changes in the presence of the inhibitor.
VI. Why does the apparent Km decrease in the presence of an uncompetitive inhibitor.
a. The binding of inhibitor to the ES complex to form EIS removes some of the ES. By LeChatelier’s principle, this
will tend to force the reaction to the right forming more ES.
i. By stimulating the binding of E and S in this manner, the graph will show that the Km is reduced.
VII. Suicide substrate – irreversibly binds to the active site permanently inactivating the enzyme
a. Important b/c they are used as potent drugs to knock out an enzyme and used to study enzyme kinetics w/ a focus on
interactions at the active site.
VIII. If we made a Lineweaver–Burk plot of an irreversible inhibitor, which type of reversible inhibition would it be most likely to
resemble?
a. Pure noncompetitive
IX. See question 60 and 61
X. Is it good (or bad) that enzymes can be reversibly inhibited? Why?
a. VERY good in the case of noncompetitive inhibitors.
i. Much of the metabolic control depends on feedback inhibition by downstream noncompetitive inhibitors.
The question is perhaps moot in the case of competitive inhibitors which are much less commonly
encountered in vivo.
1. CI can be seen in antibiotics ood for sick and bad for bacteria

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