Enzymes

• Catalysts for biological reactions

• Most are proteins
• Lower the activation energy
• Increase the rate of reaction
• Activity lost if denatured
• May be simple proteins
• May contain cofactors such as metal ions or
organic (vitamins)
Enzymes
 Enzyme Active Site
• Amino acid side chains interact, metal ions,
• Various types of polar, non-polar, ionic interactions
 Enzymes
• Lock and Key Analogy: lock = enzyme, key = substrate.
 Induced Fit Theory
• Enzyme is not rigid, changes shape with substrate.
 Enzymes - Cofactors
• Coenzymes (contain a vitamin) or metal ion
 Enzymes - Cofactors
• Metal ions present in trace amounts
 Enzyme Cofactors
• Coenzyme - non protein organic, maybe a vitamin.
Enzymes
 Enzymes - Activity
• Temperature and pH effect enzyme action
 Enzymes - Activity
• Enzyme and substrate concentrations
 Enzymes - Inhibitors
• Nonspecific - denaturation, pH, temperature
 Enzyme Inhibition
• Competitive
• Uncompetitive
• Mixed (Non-competitive)
 Enzyme Inhibitors
• Noncompetitive
Mixed (Non-competitive)
 Enzyme Inhibitors
• Competive - mimic substrate, may block active
site, but may dislodge it.
Enzyme Inhibitors
Competitive Inhibition
Competitive as M-M
 Enzyme Inhibitors
• Irreversible - covalently binds to active site
Uncompetitive
Effects of Inhibitors on the Parameters of the
Michaelis–Menten Equationa
 Enzyme Inhibitors
• Regulator or feedback - used to control a sequence
of reactions - reaction product may block initial
enzyme.
 Name of Enzymes
• End in –ase
• Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
• Describes function of enzyme
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
• Common names of digestion enzymes still use –in
pepsin, trypsin
 Classification of Enzymes
Class Reactions catalyzed
• Oxidoreductoases oxidation-reduction
• Transferases transfer group of atoms
• Hydrolases hydrolysis
• Lyases add/remove atoms
to/from a double bond
• Isomerases rearrange atoms
• Ligases combine molecules
using ATP
 Examples of Classification of
Enzymes
• Oxidoreductoases
oxidases - oxidize ,reductases – reduce
• Transferases
transaminases – transfer amino groups
kinases – transfer phosphate groups
• Hydrolases
proteases - hydrolyze peptide bonds
lipases – hydrolyze lipid ester bonds
• Lyases
carboxylases – add CO2
hydrolases – add H2O
 Learning Check E1
Match the type of reaction with the enzymes:
(1) aminase (2) dehydrogenase
(3) Isomerase (4) synthetase
A. Converts a cis-fatty acid to trans.
B. Removes 2 H atoms to form double bond
C. Combine two molecules using ATP
D. Adds NH3
 Solution E1
Match the type of reaction with the enzymes:
(1) aminase (2) dehydrogenase
(3) Isomerase (4) synthetase
A. 3 Converts a cis-fatty acid to trans.
B. 2 Removes 2 H atoms to form double bond
C. 4 Combine two molecules using ATP
D. 1 Adds NH3
 Enzyme Action:
Lock and Key Model
• An enzyme binds a substrate in a region called the
active site
• Only certain substrates can fit the active site
• Amino acid R groups in the active site help
substrate bind
• Enzyme-substrate complex forms
• Substrate reacts to form product
• Product is released
 Lock and Key Model

P
+ S +
S
P

E + S ES complex E + P
 Enzyme Action:
Induced Fit Model
• Enzyme structure flexible, not rigid
• Enzyme and active site adjust shape to bind
substrate
• Increases range of substrate specificity
• Shape changes also improve catalysis during
reaction
 Enzyme Action:
Induced Fit Model

P
S
S
P

E + S ES complex E + P
 Learning Check E2
A. The active site is
(1) the enzyme
(2) a section of the enzyme
(3) the substrate

B. In the induced fit model, the shape of the

enzyme when substrate binds
(1) Stays the same
(2) adapts to the shape of the substrate
 Solution E2
A. The active site is
(2) a section of the enzyme

B. In the induced fit model, the shape of the

enzyme when substrate binds
(2) adapts to the shape of the substrate
Factors Affecting Enzyme Action:
Temperature
• Little activity at low temperature
• Rate increases with temperature
• Most active at optimum temperatures (usually
37°C in humans)
• Activity lost with denaturation at high
temperatures
 Factors Affecting Enzyme Action
Optimum temperature

Reaction
Rate

Low High
Temperature
 Factors Affecting Enzyme Action:
Substrate Concentration
• Increasing substrate concentration increases
the rate of reaction (enzyme concentration is
constant)
• Maximum activity reached when all of enzyme
combines with substrate
 Factors Affecting Enzyme Action

Maximum activity

Reaction
Rate

substrate concentration
Factors Affecting Enzyme Action:
pH
• Maximum activity at optimum pH
• R groups of amino acids have proper charge
• Tertiary structure of enzyme is correct
• Narrow range of activity
• Most lose activity in low or high pH
 Factors Affecting Enzyme Action

Reaction
Rate
Optimum pH

3 5 7 9 11

pH
 Learning Check E3
Sucrase has an optimum temperature of 37°C and
an optimum pH of 6.2. Determine the effect of the
following on its rate of reaction
(1) no change (2) increase (3) decrease
A. Increasing the concentration of sucrose
B. Changing the pH to 4
C. Running the reaction at 70°C
 Solution E3
Sucrase has an optimum temperature of 37°C and
an optimum pH of 6.2. Determine the effect of the
following on its rate of reaction
(1) no change (2) increase (3) decrease
A. 2, 1 Increasing the concentration of sucrose
B. 3 Changing the pH to 4
C. 3 Running the reaction at 70°C
 Enzyme Inhibition
Inhibitors
• cause a loss of catalytic activity
• Change the protein structure of an enzyme
• May be competitive or noncompetitive
• Some effects are irreversible
 Competitive Inhibition
A competitive inhibitor
• Has a structure similar to substrate
• Occupies active site
• Competes with substrate for active
site
• Has effect reversed by increasing
substrate concentration
 Noncompetitive Inhibition
A noncompetitive inhibitor
• Does not have a structure like substrate
• Binds to the enzyme but not active site
• Changes the shape of enzyme and active site
• Substrate cannot fit altered active site
• No reaction occurs
• Effect is not reversed by adding substrate
 Learning Check E4
Identify each statement as describing an inhibitor
that is
(1) Competitive (2) Noncompetitive

A. Increasing substrate reverses inhibition

B. Binds to enzyme, not active site
C. Structure is similar to substrate
D. Inhibition is not reversed with substrate
 Solution E4
Identify each statement as describing an inhibitor
that is
(1) Competitive (2) Noncompetitive

A. 1 Increasing substrate reverses inhibition

B. 2 Binds to enzyme, not active site
C. 1 Structure is similar to substrate
D. 2 Inhibition is not reversed with substrate
 Many drugs are enzyme
inhibitors
Drug target use page
Aricept acetylcholinesterase Alzheimer disease 130
Aspirin COX-1, COX-2 inflammation 515
L-738,317 HIV protease HIV 160
Lovastatin HMG CoA reductase high cholesterol 523
Methotrexate dihydrofolate reductase cancer 583
Penicillin bacterial transpeptidase antibiotic 254
Caffeine cAMP phosphodiesterase stimulant 296
 Types of enzyme inhibitors
• Reversible
– Competitive
– Uncompetitive
– Noncompetitive
• Irreversible
 Competitive inhibition
• Raises apparent Km
• Vmax unaffected

Figs. 5.8, 5.9

 Competitive inhibitors mimic
substrates

Rational Drug Design

substrate Purine nucleoside phosphorylase Potent inhibitor

Fig. 5.13
 Uncompetitive inhibition
• Lowers apparent Km
• Lowers Vmax
• Vmax / Km unchanged

Figs. 5.8, 5.11

 Noncompetitive inhibition
• apparent Km unaffected
• Lowers Vmax

Figs. 5.8, 5.12

 Irreversible inhibition
• Forms stable covalent bond with enzyme
inactivating it
• Affinity labels are active-site-specific
chymotrypsin

F-
H+

DFP
An organophosphate

Fig. 5.15
 Enzyme activity is regulated in
multiple ways
• Slow
– Synthesis (more later…)
– Degradation (more later…)
• Rapid
– Reversible covalent modification
– Noncovalent allosteric regulation
 Characteristics of an allosteric
enzyme
• Activity is changed by
metabolic inhibitors or
activators
• Allosteric modulators
bind noncovalently
• Possesses quaternary
structure
• Demonstrates
sigmoidal kinetics
Fig. 5.21
 Case study: E. coli
Phosphofructokinase
• Catalyzes early step in
PFK tetramer
glycolysis
– ATP-generating pathway
for glucose degradation Active site

• Tetramer
• Substrates
– F6P
– ATP
• Allosteric effectors
– PEP
– ADP

ADP
Fig. 5.20 PFK monomer
 Feedback inhibition
Glucose

• PEP inhibition of PFK

Fructose 6-phosphate
conserves energy and
PFK
material when end
Fructose 1,6-bisphosphate
product is abundant
-

Phosphoenolpyruvate
(PEP)
 Allosteric activation
• When energy is low, ADP
Glucose
stimulates PFK leading to
increased glycolysis and more
ATP (energy)
Fructose 6-phosphate
+
ADP PFK

Fructose 1,6-bisphosphate

ATP
(work)

Phosphoenolpyruvate
(PEP)
Fig. 5.19
 Models for allosteric regulation
• Remember the R and T states of
Hemoglobin
• Allosteric inhibitors favor the T state
• Allosteric activators and substrates favor the
R state
 Two models for
cooperativity
• Concerted
– Symmetry driven- all
R or All T. Substrate
stabilizes R.
• Sequential
– Ligand induced –
neighboring
unoccupied subunits
are variably affected

Fig. 5.22
Enzyme Inhibitors
 Drugs as Enzyme Inhibitors
• Drug mimics some natural chemical to block enzyme.
 Regulation of Enzyme Activity
• By control of enzyme levels
• By control of enzyme activity
• Allosteric Mechanisms
Aspartate transcarbamylase
Feedback Inhibition