BISC 312, Lecture 6:

Enzymes
Chapter 8
 Enzyme Concepts

Enzymes are powerful catalysts with high substrate specificity

DO increase the rate of biochemical reactions
DO NOT alter the equilibrium of a reaction
DO NOT affect the energetics of the reaction (ΔG)
ATP hydrolysis can aid in the energetics (net ΔG is negative)

Enzymes often require co-factors (coenzymes or metals)

for catalytic activity

 Change in free energy (ΔG) is a valuable thermodynamic parameter to

estimate if a reaction can take place and evaluate the energy cost of the
reaction.

A reaction doesn’t proceed just because there is a negative ΔG. It’s

important that this is true or reactions could proceed uncontrollably.
 Enzymes: Basics

• Enzymes are generally proteins

Sometimes RNA (ribozymes)
Trypsin Group I Ribozyme
domain
• Act as catalysts for biochemical reactions
- Form complexes by binding a substrate at an active site (provide unique
environment for catalysis)
E+S ES
-Binding is HIGHLY SPECIFIC, usually noncovalent interactions
-Enzymes are not chemically altered by the reaction
-Enzymes do not change the equilibrium constant Keq of the reaction,
it will simply approach equilibrium more quickly

Enzymes INCREASE THE RATE of a biochemical reaction by lowering the

activation energy to go from substrate (S) to product (P)
 Catalytic substrate specificity of enzymes
1- substrate they recognize
Enzymes are EXTREMELY specific:
2- reactions they catalyze

e.g. Proteases: catalyze the hydrolysis of peptide bonds in proteins More than 550
different
proteases in the
human body

Thrombin: Proteolytic enzyme involved in •

blood clotting cascade

Recognizes a specific sequence of amino acids:

•
Leu-Val-Pro-Arg-Gly-Ser
Very specialized & highly specific (only where blood needs to clot)
You don’t need to memorize these, they are just FYI as we will see them in metabolic pathways.
 Chemical reaction rates
Rate: velocity of reaction (V)
In other words: the quantity of material that disappear (S) or appear (P) by unit of time
Rate units: concentration per second or M s-1

• Lets consider the simple chemical reaction A B

Rate V = -ΔA/Δt = ΔB/Δt

If we focus only on the disappearance of S, the rate of reaction is proportional to [S] as follows:
V = k[A] (first order reaction) k is the rate constant of the reaction. Units in s-1
Enzymatic reaction rates in reversible reactions
As product accumulates, the reverse reaction becomes important.

k1
A B
k-1

When forward reaction is equal to reverse reaction, we are at equilibrium

[B] [k1]
k1[A] = k-1[B] K= = [k ] equilibrium constant
[A] -1
 Understanding how enzymes work: kinetics
Enzymes alter the rate of a biochemical reaction NOT its equilibrium

Reaction has reach equilibrium

• Enzymes accelerates the time it takes to reach

equilibrium, the rate in both directions.
• Enzymes have no effect on the equilibrium
itself

There is a relationship between reaction rate and equilibrium constant:

kF kF: rate constant of forward reaction
kF[S] = kR[P] S P kR: rate constant of reverse reaction
kR

Keq = [P] = kF 10-4 = 100 = K eq

[S] kR Example: kF = 10-4 s-1 and kR = 10-6 s-1 10-6
 Second order reactions
• Lets consider the more complex chemical reaction A+B C
The rate of reaction is now proportional to both [A] and [B] as follows:
V = k[A][B] (second order reaction) k is the rate constant of the reaction. Units in M-1s-1

Enzyme-Substrate reactions are generally second-order and can be multi-step reactions

that are limited by the slowest step in the reaction, the rate-limiting step.
 Understanding how enzymes work: energetics
Whether a reaction takes place at all and the degree to which enzymes accelerate the
reaction depends on: energy difference between reactant and products
FREE ENERGY (G) is a measure of useful energy, that is energy capable of doing work

Difference in FREE ENERGY (ΔG) is a useful thermodynamic value to estimate if a

reaction can take place spontaneously or not.

ΔG = Gproducts (final state) – Greactants (initial state)

Reactants Products
• If ΔG < 0 (negative value): reaction proceed to the right spontaneously (exergonic)
• If ΔG = 0 : reaction at equilibrium and no net changes
• If ΔG > 0 (positive value): reaction (left to right) does not take place spontaneously
Note: however reaction right to left is spontaneous (endergonic reaction)

• Spontaneous reaction = release of energy (exergonic) no need for extra input of energy
• Non-spontaneous reaction = need for extra input of energy if you want the reaction to go
left to right (endergonic)
 Transition State and Reaction Rate

 Enzymes increase the rate of reactions by decreasing the activation

energy barrier ΔG‡ of the reaction
• The lower the activation energy (ΔG‡) the higher the rate constant (k)
• Small changes in activation energy (ΔG‡) leads to large change in rate constant (k)
 How can we increase a reaction rate?

Increase the rate constant by:

a- Increase the
temperature
b- decrease the activation
energy (ΔG‡)

In biological systems, where temperature

changes are not tolerated, only a small
percentage of molecules will have the free
energy to attain the transition state.

Enzymes increase the rate of reaction by

decreasing the activation energy, ΔG‡, thus
increases the number of molecules that can
achieve the transition state.
 How do enzymes affect lower the activation energy?
Enzymes facilitate the formation of a transition state:

S P S S‡ P
Transition state
Transition State (S‡):
• A particular chemical configuration of the substrate as it evolves toward
becoming the product during reaction
• Very short lived (transient) chemical state
• Usually highest free energy peak of free energy diagram
 Mechanistically how do enzymes decrease ΔG‡ ?

They bind the substrate and provide an environment

that favors the formation of the transition state rather
than original form (S)

A transition state tightly bound to the enzyme is at

lower energy level than the transition state in free
solution in an uncatalyzed reaction, primarily by
decreasing ΔH, enthalpy.

Therefore, the energy involved to adopt the transition

state is lower than without the enzyme.
Intermediate states of reactions

Sometimes the reaction

pathway is altered by
enzymes to form an
intermediate state that
has a lower free
energy than a
transition state in an
uncatalyzed reaction.

ie. the chemical structure

of the catalyzed transition
state looks different
Models for binding and catalysis
 Enzymes form a complex with their substrates in active site

• 3D catalytic site/pocket
• Multiple weak interactions between enzyme and substrate
• Strong binding yet flexible

 Enzymes use induced fit mechanisms (vs. lock

and key) to bind substrates with high specificity
and to increase the rate of biochemical reactions.
Both the enzyme and substrate change
conformations to allow formation of the
transition state.
Explains both binding specificity and catalysis.
 Binding of substrate to enzyme
Physical evidence for enzyme-substrate interaction: ES complex (Hexokinase)

The shape of active catalytic pocket is flexible and can change

conformation after binding of the substrate to become sterically and
chemically complementary to the substrate (induced fit)

• “Breathing” of the enzyme is

key for highly specific binding of
the substrate…
• … but also for strong binding to
the transition state…
• … and for release of the product
 Different ways enzymes enhance the rate of
reactions:

1. Preferential binding to transition state through noncovalent interactions (electrostatic

catalysis, makes ΔH more favorable)

2. Distortion of substrate or active site, promoting change to transition state (induced fit)

3. Binding of substrates to optimize proximity and orientation (makes ΔS more favorable

by decreasing initial entropy of reactants).

4. Altering reaction pathway to include alternative intermediates

(occurs often in covalent catalysis)

5. Other mechanisms: General Acid-Base catalysis (GABC) or Metal Ion catalysis

 Cofactors
Cofactors are small molecules that enzymes need to execute biochemical reactions

apoenzyme + cofactor holoenzyme

• Cofactors are either coenzymes or metals
Coenzymes can be: • weakly bound : cosubstrates associate or dissociate during reaction
• tightly bound to enzymes: prosthetic groups

Coenzyme (cosubstrate) Metals

NADH
Zn2+
Lactate
dehydrogenase
(motif) Carbonic anhydrase
Box 7.2
Princ. of Biochem. 5th edition
2012, Pearson
 Coenzyme function in catalysis: NAD+ as electron
acceptor, an oxidizing agent

accepts a hydride ion and converts alcohols

to ketones or aldehydes

Nicotinamide adenine
dinucleotide (NAD+)
is derived from the
vitamin, Niacin
 Please watch these videos:
The specifics of deriving the M-M equation
won’t be tested, but it’s good to know where
these values come from. You WILL need to
know how to interpret and apply values like Km
and Vmax

• https://youtu.be/X_YXTWU2maY
• https://youtu.be/7u2MkbsE_dw
 Studying enzymatic reaction rates
A good way to study enzymatic reaction rates and determine if an enzyme
follows M-M kinetics is to follow the disappearance of the substrate (S) over
time for different concentrations of substrate [S]
kF
S P
kR

Recall: V = -ΔS/Δt = kF [S]

Notice:
• V slows down as S is converted to P
• Reverse reaction becomes significant as
P accumulates, leading to no change in
ΔS/Δt at long time: Equilibrium VF =VR
no net disappearance of S

initial rate vs. [S] When plotting [S] over time (t) the initial
slope of the curve is the rate V0

V0 is measured at very early time point. Before [S]

decreases
 Interpreting the Michaelis-Menten curve

Vmax[S]
V0 = -----------
3 KM + [S]
Observations

1. At very low [S], [S] <<< KM, then

Vmax[S]
2 V0 = ------------
KM
Vmax
2. At [S] = KM, V0 = -----------
2
1 3. At very high [S], [S] >>> KM,
then V0 = Vmax and rate is
independent of [S]
 How to determine KM and Vmax accurately?
Reciprocal plot of Michaelis-Menton curve
Vmax[S] 1 K 1 1
V0 = ------------- reciprocal ---- = ----M * ---- + ----
KM + [S] Vo Vmax [S] Vmax

Resembles: y = mx + b
• x-intercept: -1/KM
• y-intercept: 1/Vmax
• slope: KM/Vmax

Remember that KM is a concentration

value. It is always positive
 Enzymatic inhibitors
Inhibitors: Small compounds that affect the activity of enzymes
2 types of inhibition: :
• Reversible (on-off binding, noncovalent)
• Irreversible (very tight binding to enzyme,
covalent)
Amongst reversible inhibitors 3 modes of action:

mixed
d

Competitive Uncompetitive Mixed

Increase KM but no Decrease both KM Decrease Vmax but
effects on Vmax and Vmax increases KM
(non-comp = no
change in Km)
 Competitive inhibition

• Enzyme binds S or I (not both)

• Enzyme can be freed from I by
increasing [S]
• KM is increased (need more S to
Ki: dissociation reach Vmax/2)
constant of inhibitor • Vmax is unchanged
Ki = [E][I]/[EI]
Uncompetitive inhibition

• I binds to ES complex only

• I binding possible only after S binding
• ESI complex can not make P
• Vmax can not be attained and is lower
• KM also lower (proportionally to Vmax)
need less S to reach Vmax/2.
• High [S] does not overcome inhibition
Mixed inhibition
• E can bind I and S at the same time
• ESI complex can not make P
• I must dissociate for catalysis to occur
• Vmax is lower
• KM is increased or same (non-comp)
• High [S] does not overcome inhibition
 Irreversible inhibition
Combine covalently with the enzymes to inactivate them

Many are transition state analogs. Similar to the transition state in structure. Binds
very strongly to enzyme (tighter binding than substrate).

Example: DFP binds to active site serine of enzyme,

acetylcholinesterase, which is necessary for nerve conduction
and causes paralysis of vital functions.

Very toxic substance

Useful:
• To understand catalytic mechanism of an enzyme
• As highly specific inhibitors
 FYI: Many pharmaceutical drugs are inhibitors

• Penicillin: Irreversible Inhibitor. Transition state analog. Inhibits Glycopeptide

transpeptidase, that normally form bacterial cell walls by cross-linking peptides.
Antibiotic functions by inhibiting cell wall synthesis.

• Statins: Competitive inhibitors of HMG-CoA reductase involved in cholesterol

synthesis. Mevacor (Lovastatin) structure similar to HMG-CoA reductase
substrate. Cholesterol reducing drugs.

• Aspirin: Irreversible covalent inhibitor of Prostaglandin H2 synthase involved in

prostaglandin synthesis (transmission of pain information, inflammation)
Anti-inflammatory action aspirin by acetylation of a serine residue in channel to
reach active site.
 Regulation of Enzyme Activity

Enzymes are often in assembly lines to carry out

sequential steps in a larger pathway. However, often it
doesn’t work to have all enzymes operating at peak
activity all the time.
Activity is regulated by different mechanisms:
• Substrate-level control
• Feedback Control
• Allosteric Enzymes
 Substrate-level Control

Direct interaction of substrate

and products with enzyme.
High levels of substrate can
increase reaction rate; however,
high levels of product also
Glucose-6-Phosphate, the product of the reaction
compete for binding to enzyme
catalyzed by hexokinase inhibits formation of
and inhibit formation of more
more G-6-P when high levels are reached.
product.
Product inhibition by both Competitive Inhibition
Uncompetitive inhibition.
Binds both to active site and a second site.
 Regulations at key points along enzymatic pathways
Enzymes that are subjected to tight regulation often catalyze “committed steps”
along specific metabolic pathway.

• A committed catalytic step is basically irreversible

• The product of a committed step will essentially continue down the pathway
to final product
• Targeting the first committed catalytic step along a enzymatic pathway helps
regulate the end product formation without unnecessary loss of energy

Feedback regulation (inhibition

or activation)
x
Enz4 Enz5
Enz3 D E F (final product 1)
Enz1 Enz2
A B C
Enz6
x L M N O P (final product 2)
Enz7 Enz8 Enz9 Enz10

Feedback regulation
 Allosteric Enzymes
Enzymes regulated by allosteric control (allosteric enzymes) are multi-subunits proteins

Regulation of catalytic (kcat) and binding activities generally done by inducing

significant conformational changes effected by :

• the substrate itself at catalytic sites (cooperativity between multiple

catalytic subunits). Homoallosteric effect
• By other ligands binding at sites distinct from the catalytic site to increase or
decrease enzymatic activity. Heteroallosteric effect.

Allosteric controls often involved in rapid switching between a highly active

catalytic state (R) and a less active state (T) of the enzyme

Useful for quick responses to rapid metabolic changes and needs!

 Allosteric controls (continued)
Allosteric enzymes do not follow Michaelis-Menten kinetics

Cooperative binding: binding of 1 substrate lead to increased binding affinity for

substrate and higher catalytic activity at other active sites on an allosteric enzyme

Non cooperative
enzyme • High [S]: High binding affinity
for substrate and high catalytic
rates

Allostric enzyme • Increase in [S] lead to a sharp

increase in catalytic rate (high
slope) until maximum rate is
reached.

• Low [S]: low binding affinity for

substrate and low catalytic rates
Heteroallosteric effectors regulate transition from T
to R state at a different site than the active site

Can be activators or inhibitors of the enzyme