Introductory

Biochemistry
Enzymes
What’s So Great About Enzymes?
Extracting Energy From Glucose
Burning Hydrocarbon Fuels
Nitrogen Fixation
N2 + 3H2 NH3

 Industrial process
 ~200 atmospheres and 400°C

 Bacteria
 Atmospheric pressure, ambient temperature
Digesting Breakfast

In the absence of enzymes,

digesting a meal could take:

A. 50 hours
B. 50 days
C. 50 weeks
D. 50 years
E. 50 decades
 Introductory
Biochemistry
Enzymes
What are Enzymes?
Enhancing Reaction Rates
A+B C+D

A reaction can be accelerated in two ways:

 Adding heat
 Increases the number of reactants with sufficient
energy to overcome activation energy barrier.
 Adding a catalyst
 Decreases the activation energy barrier but does not
react.
Enzymes are Proteins
 With few exceptions, enzymes are proteins.

 1º, 2º, 3º, 4º structures.

 Typically globular proteins.

 Structure is governed/determined by the same

forces.
Enzymes are Catalysts
 Accelerate reaction rates
 Regenerated at the end of the reaction
 106 to 1020 fold increases in reaction rates
 Highly specific
 A+BC+D
 No side reactions
Enzyme Nomenclature
 Typically end in –ase
 Name often describes the process:
 Substrate name (or product name)
 Chemical Reaction
 Examples:
 Citrate synthase
 Alcohol dehydrogenase
 Pyruvate decarboxylase
Enzymes are Regulated
 Distinguishes enzymes from non-biological
catalysts
 As proteins, enzyme structures are (to an
extent) flexible
 Changing the shape of an enzyme changes
its function
 Introductory
Biochemistry
Enzymes
How Do Enzymes Work?
Which of the following
reactions is faster?

A+B
A. A + B → C + D
B. A + B → E + F
C+D
G C. I can’t tell from this
E+F diagram.

Initial state Final state

Reaction Coordinate
In all systems, biological or otherwise, a reaction
will proceed only if the free energy of the products
is less than the free energy of the reactants.

Grxn = GP - GR

When Grxn is negative:

Reaction is exergonic and

“thermodynamically favorable”
(spontaneous)

Thermodynamically favorable reactions DO NOT NECESSARILY

proceed at measurable rates
The Speed of a Favourable Biochemical
Reaction is Determined by the Size of the
Activation Energy Barrier

AB + C [A•B•C] A + BC
Transition state (TS)
Highest free energy
The Speed of a Favourable Biochemical
Reaction is Determined by the Size of the
Activation Energy Barrier

G‡ = GTS - GR

G‡
Enzymes Reduce the Free Energy
of the Transition State
Enzymes do not affect
the free-energy change
(DG) of the reaction!
Which of the following statements is TRUE
regarding the free energy diagrams below?

A B

Reactants

Products

A. The free energy change of reaction B has a larger magnitude than

that of reaction A.
B. Reaction B is faster than reaction A.
C. Both statements are true.
 How Do Enzymes Reduce the Free
Energy of the Transition State?

1. Removing substrates
from aqueous solution
(desolvation).
2. Proximity and orientation
effects.
3. Taking part in the
reaction mechanism.
4. Stabilizing the transition
state.
Active Sites
 Region of enzyme where catalysis occurs
 Usually only a small portion of the protein
 Key amino acids are located in active site
 Binding
 Catalysis
 Determines affinity, specificity, rate
 Complementary to substrate/transition state
 Shape, hydrophobic interaction, H-bonds, ion pairs
The Active Site in Lysozyme
The active site is a
3-D cleft/crevice within
the 3-D shape of the
protein
Complementarity in Substrate
Binding
Design of the active site
contributes to:
Interactions
between substrate
• Affinity and enzymes?
• Specificity

Lock and Key Model

Which of the following groups of amino acid side chains is
MOST LIKELY to be positioned into the active site of an
enzyme that binds glucose as a substrate?

A. Gln, Asn, Ser

B. Val, Leu, Ile
C. Trp, Phe, Ile
D. Val, Glu, Lys
E. Cys, Met, Pro
Desolvation (Exclusion of Water)
 Exclusion of water provides three advantages:
 Removal of water shell accelerates reactions.
 Enhances polar interactions (H-bonds, ion pairs)
 Prevents side reactions.

 “Induced Fit”
 Some enzymes change shape when substrate
binds
 Closes off active site (excludes more water).
 Brings catalytic/reactive groups together.
Desolvation-Removing substrates from aqueous
solution: e.g. Substrate Binding by Hexokinase

Induced fit -some enzymes show a pronounced conformation change upon

binding of substrate
Induced Fit Model: Active site changes shape as substrate(s) bind.
Proximity and Orientation
 Chemical reactions only occur if substrates
come together in the right orientation to react

 Active sites bind substrates close to each

other (proximity) and in the correct geometry
(orientation)
2. Proximity and Orientation
Effects in Catalysis

May account for a thousand-fold increase in reaction rates

All enzymes do this
Participation in Reactions
 Some enzymes participate in the reactions by
positioning functional groups near the
substrates in the active site. These groups
may function as:
 Acid/base catalysts
 Covalent catalysis
 Metal ion catalysis

 This may be achieved through either amino

acids or cofactors (or both).
Amino Acid Side Chains in Acid-Base
Catalysis

 These groups can

act as acid or base
catalysts,
depending on their
state of protonation.
 Amino Acid Side Chains in
Nucleophilic Catalysis (covalent
catalysis)

 In their de-protonated forms, these groups can act as nucleophiles.

The Classification of Cofactors
Molecules/compounds that may enhance the reactive potential of
polypeptides by providing new reactive functional groups

COFACTORS

Coenzymes Metal Ions

(organic molecules) (e.g. Fe, Zn, Cu, Na, Mg, K)

Prosthetic Groups Loosely Bound

Cosubstrates Prosthetic Groups

(e.g. NAD+) (e.g. FAD)

These coenzymes need to be

regenerated after reactions
Apoenzyme vs. holoenzyme
 For enzymes that contain a prosthetic group:
 When the polypeptide is combined with the prosthetic
group to form the functional tertiary structure it can be
referred to as the holoenzyme.
 Without the prosthetic group, the polypeptide can be
referred to as the apoenzyme.

 Holoprotein and apoprotein can be used respectively to refer to

non-enzymatic proteins with or without prosthetic groups (e.g.
apomyoglobin is the myoglobin polypeptide chain without heme
associated).
Transition State Stabilization
 Binding the transition state will aid in lowering DG‡.
 Parts of the protein interact with the unstable transition state.
 Enzyme active sites bind the transition state better than they bind
the substrate.
substrate transition state products

“Preferential Transition State Stabilization”

The more tightly an enzyme
binds the transition state relative to
the substrate the greater the catalytic
activity of the enzyme

Transition state analogs are POTENT inhibitors

of many enzymes
• bind to enzyme with higher affinity compared to substrate
• rational basis for drug design
Chymotrypsin
(Don’t need to memorize, just exemplifies mechanisms discussed)

Formed by backbone NH groups of residues 193 and 195, the oxyanion hole is partially
positive and able to form hydrogen bonding interactions with a negatively-charged
oxygen that develops during the reaction.
Which of the following is NOT a way that
enzymes increase reaction rates?

A. By removing reactants from the aqueous

environment.
B. By stabilizing a high-energy transition state.
C. By lowering the value of DGreaction.
D. By providing specific reactive groups in the
active site.
E. All of these are used by enzymes to
increase reaction rates.
 Introductory
Biochemistry
Enzymes
Regulating Enzyme Activity
Reaction Velocity Vs.
Concentration of Substrate

[Enzyme] is fixed
Enzyme kinetics
 V0: Initial velocity (rate of product formation).
 Vmax: Maximum rate of product formation.
 Km: Michaelis constant
 Analogous to Kd in ligand binding
 Reflects “affinity” of enzyme for substrate
 Smaller as affinity increases
 V0 = 50% Vmax when [substrate] = Km
Reaction Velocity Vs.
Concentration of Substrate

A B

The enzyme has a higher affinity

for which substrate?

A
B
KM

[Enzyme] is fixed
In graph shown below which of the following statements is
TRUE concerning the “plateau” region of the curve?

A. All the available substrate has been converted to product.

B. The enzyme active site is becoming saturated.
C. The enzyme is in the tense state.
D. A and C
E. A and B
Some Mechanisms for the
Regulation of Enzyme Activity

 Competitive inhibition.
 Allostery. These affect the
intrinsic activity
 Reversible covalent modification. of the enzyme.

 Ionic signals (e.g. Ca2+ ions).

 Regulation of gene expression. Do not affect

the intrinsic
 Changes in subcellular localization. activity of the
enzyme.
Competitive Inhibitors
 Substances that bind reversibly in the active site.
 Resemble the substrate or transition state but do not
react.

 Physically blocks active site (prevent substrate binding)

 Fewer active sites available
 Lower reaction rates (inhibited, curves shift right)
 Apparent increase in Km

 Increasing substrate concentration will overcome

inhibition
 No change in Vmax
Competitive Inhibition of
Enzyme Activity
Inhibitors are similar to
the substrate in shape and
size but differ chemically
in such a way that they
S cannot react
S
S S S

S
S S

S
Competitive inhibitors decrease the
apparent affinity (increase KM) for
enzyme and substrate

apparent KM
with inhibitor
KM without
inhibitor

Transition state analogs make better competitive inhibitors

than substrate analogs. WHY?
Allosteric Enzymes
 Many enzymes are oligomeric (multi subunit).
 Like hemoglobin, enzyme activity may be
cooperative.
 Sigmoidal relationship between substrate and reaction velocity.
 Reflects different states (geometries) for the active site.
 Change between low activity (T) and high activity (R) states.

 Compounds besides the substrate may affect the

equilibrium between T and R states.
 Allosteric effectors (positive/negative; activators/inhibitors).
 Very common in regulation.
Allosteric enzymes show a sigmoidal
relationship between reaction velocity and
substrate concentration

Homoallostery
(positive)
An allosteric enzyme’s catalytic activity is
modulated by the noncovalent binding of specific
molecules at a site other than the active site
(heteroallostery)

Positive
Homoallostery

/reduced
Negative
Heteroallostery
Allosteric enzymes have two states, T ( tense,
low activity) and R (relaxed, high activity)

ALLOSTERIC
Activator
favors R state

ALLOSTERIC
Inhibitor
favors T state
Allosteric Enzymes may be Inhibited
or Activated
This graph represents the rates of two
distinct enzyme-catalyzed reactions.
Which of the following statements is
FALSE?
1

A. Enzyme 1 is non-allosteric.
B. Enzyme 2 is allosteric.
C. The substrate for enzyme 2 is a homoallosteric
activator of enzyme 2 .
D. A heteroallosteric activator of enzyme 2 would
cause the blue dotted curve to assume the shape
of the red curve .
How might the (reversible) attachment of
an inorganic phosphate group to a
hydroxyl-containing amino acid side chain
(Ser, Thr or Tyr) alter the activity of an
enzyme?
A. The phosphate group blocks the
active site and prevents the substrate
from binding.
B. Addition of the phosphate group
changes the shape of the enzyme.
C. The binding affinity of the substrate is
lowered because of electrostatic
repulsion.
D. Addition of the phosphate group
causes the enzyme to dissociate into
its separate subunits which are all in
the R state.
E. All of the above
Reversible Covalent Modification
 Covalent modification of an amino acid residue changes
the 3° structure of a polypeptide.

 Phosphorylation is the most common type of reversible

covalent modification.
 Ser/Thr/Tyr –OH becomes phosphorylated.
 Increases size, polarity, and charge significantly.

 May increase or decrease activity of the target enzyme

Phosphorylation of enzymes
changes the tertiary structure

May also lead to decrease in activity or inactivation.

The Reversible
Phosphorylation of Enzymes
Protein kinases
Protein kinases catalyze the
phosphorylation of proteins.

Protein phosphatases catalyze

the dephosphorylation of
E1 E1 proteins by hydrolysis.

Unphosphorylated Phosphorylated Both kinases and phosphatases

protein protein
are enzymes, often regulated
themselves.
H2O
Protein phosphatases
Pi
 Ser /Tyr /Thr -OH

ATP Pi

Protein Kinase Protein Phosphatase

ADP H2O

O
‫װ‬
Ser /Tyr /Thr -O-P-O-
I
O-

Other amino acids can be phosphorylated

(e.g. Asp, Glu and His) but not typically part of
regulation (e.g. Asp in the Na+K+ ATPase).
Which of the following
statements about protein kinases
is FALSE?

A. Protein kinases are proteins.

B. Protein kinases are enzymes.
C. The substrate of a protein kinase is a protein.
D. One protein kinase may be a substrate for another
protein kinase.
E. None of these statements is false.
Which of the following methods of altering
enzyme activity is irreversible?

A. Binding of transition state analogs to enzymes.

B. Binding of competitive inhibitors to enzymes.
C. Binding of allosteric effectors to enzymes.
D. Phosphorylation of enzymes.
E. None of the above.
Which of the following graphs is most consistent with the
results of reaction rate vs [S] for an allosteric enzyme in the
absence and presence of an allosteric inhibitor?

A. 1
B. 2
C. 3
D. 4
E. None of the above
graphs