Principles of Enzymes

(Plant Biochemistry)
 Enzymes
• What are enzymes?

Answer:

1. Proteins: most enzymes are proteins, primarily

tertiary and quaternary structures.

2. Catalyst: chemical agent that accelerates a

reaction without being permanently
changed in the process.
 Enzymes
3. Selective: enzymes are specific for which they
will catalyze (Specificity - depends upon
3D shape).

4. Recycled: enzymes are reusable.

Binding Catalysis Release

Enzyme Substrate Active Site Product

 Enzymes
• The turnover number is generally 1-10,000 molecules per
second.
 Enzymes
• How do enzymes work?
Answer:

• Enzymes speed up the cell’s chemical reactions by

lowering the free energy of activation.
Enzymes
 Enzyme Structure

• Apoenzyme: protein
• Cofactor: Nonprotein component
– Coenzyme: Organic cofactor
• Holoenzyme: Apoenzyme + cofactor
Enzymes
 Substrate

• The substance (reactant) an enzyme acts on.

Enzyme

Substrate
 Active Site
• A restricted region of an enzyme molecule which
binds to the substrate.

Substrate

Enzyme

Active Site
 Induced Fit
• A change in the configuration of an enzyme’s active
site (H and ionic bonds are involved).
• Induced by the substrate.

Active Site
substrate

Enzyme

induced fit
 Enzyme Classes/Reactions
1. Oxidoreductases (EC 1). Act on many chemical
groupings to add or remove hydrogen atoms.

2. Transferases (EC 2). Transfer functional groups

between donor and acceptor molecules (e.g. kinases that
transfer phosphate from ATP to other molecules).
X-Y + Z = X + Z-Y X-Y + Z-H = X-H + Z-Y.
1. Hydrolases (EC 3). Add water across a bond,
hydrolyzing it.
hydrolytic cleavage of C-O, C-N, C-C
 Enzyme Classes/Reactions

4. Lyases (EC 4). Add H2O, NH2, or CO2 across double

bonds, or remove these elements to produce double
bonds.
Lyases are enzymes cleaving C-C, C-O, C-N
5. Isomerases (EC 5). Carry out stereochemical, geometric
and other types of isomerization.

6. Ligases (EC 6). Catalyze reactions in which two

chemical groups are joined (ligated) with the use of
energy from ATP.
 FORMAT OF NUMBER
(http://www.ebi.ac.uk/pdbe-srv/PDBeXplore/enzyme/)

Every enzyme code consists of the letters "EC" followed by four numbers
separated by periods. Those numbers represent a progressively finer
classification of the enzyme.

For example, the tripeptide aminopeptidases have the code "EC 3.4.11.4",
whose components indicate the following groups of enzymes:

EC 3 enzymes are hydrolases (enzymes that use water to break up some other
molecule)
EC 3.4 are hydrolases that act on peptide bonds
EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid
from a polypeptide
EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide
 The Enzyme Commission number (EC number)
The enzyme nomenclature scheme was developed starting in 1955, when the
International Congress of Biochemistry in Brussels set up an Enzyme Commission.
The first version was published in 1961.

The current sixth edition, published by the International Union of Biochemistry

and Molecular Biology in 1992, contains 3196 different enzymes.
 Enzyme example(s) with trivial
GROUP REACTION CATALYZED TYPICAL REACTION
name

To catalyze oxidation/reduction
EC 1 reactions; transfer of H and O AH + B → A + BH (reduced)
Dehydrogenase, oxidase
Oxidoreductases atoms or electrons from one A + O → AO (oxidized)
substance to another

Transfer of a functional group

EC 2 from one substance to another.
AB + C → A + BC Transaminase, kinase
Transferases The group may be methyl-, acyl-,
amino- or phosphate group

EC 3 Formation of two products from

AB + H2O → AOH + BH Lipase, amylase, peptidase
Hydrolases a substrate by hydrolysis

Non-hydrolytic addition or
EC 4 removal of groups from
Lyases substrates. C-C, C-N, C-O or C-S
bonds may be cleaved
RCOCOOH → RCOH + CO2 or [x-
Decarboxylase
A-B-Y] → [A=B + X-Y]

Intramolecule rearrangement,
EC 5
i.e. isomerization changes AB → BA Isomerase, mutase
Isomerases
within a single molecule

Join together two molecules by

EC 6 synthesis of new C-O, C-S, C-N or
X + Y+ ATP → XY + ADP + Pi Synthetase
Ligases C-C bonds with simultaneous
breakdown of ATP
 Allosteric Enzymes

• Allosteric enzymes do not follow Michaelis-

Menten kinetics: Km & Vmax vary with [S].

• Velocity as a function of [S] gives a sigmoidal plot.

Lect. 13-17
 Allosteric Enzymes

Lect. 13-18
 Factors Influencing Enzyme Activity
• Enzymes can be denatured by temperature and pH
 What Affects Enzyme Activity?

• Three factors:

 Environmental Conditions

 Cofactors and Coenzymes

 Enzyme Inhibitors
 1. Environmental Conditions

• Enzymatic reactions are very specific.

The following environmental conditions affect
enzymatic reactions:
1. Temperature (extremes most dangerous):
- high temps may denature the enzyme.

2. pH (most like 6 - 8 pH - neutral)

3. Ionic concentration (salt ions)

 2. Cofactors and Coenzymes
• Inorganic substances (zinc, iron) and vitamins
(respectively) are sometimes need for proper
enzymatic activity.
• Example:
Iron must be present in the quaternary
structure - hemoglobin in order for it to
pick up oxygen.
 Factors Influencing Enzyme Activity
• Temperature
 Factors Influencing Enzyme Activity
• pH
 Factors Influencing Enzyme Activity
• Substrate concentration
 Factors Influencing the Rates of
Enzyme-Catalyzed Reactions

• Concentration of the substrate

Rate versus Concentration of Substrate Rate versus Concentration of Substrate
Most Enzymes Allosteric Enzyme
1.0

0.8

0.6
Rate

Rate
0.4

0.2

0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0

Substrate Concentration Substrate Concentration

 Normal enzyme

Zero order

First order

Follows Michaelis-Menten kinetics. Hyperbolic V vs. S plot

 Michaelis-Menten Graph
• Used to describe the rate of enzymatic reactions
• Plot of v vs. [S]
• Reaction rate (v) = Vmax
1 + Km/[S]

• Graph is seen as a curve

– 1st order region = slope is constant
– Mixed order region = slope is changing
– Zero order region = slope is approaching zero
 Vmax

½Vmax

Km
 Lineweaver-Burk Graph
• Used to describe the rate of enzymatic reactions
• Plot of 1/v versus 1/[S]
• Where 1 = Km 1 + 1
v Vmax [S] Vmax

*Graph is seen as a straight line

*Easy to find values for Vmax and Km

 Significance of Km and Vmax
• Km
– [S] at which the reaction proceeds at half its maximum
velocity
– Equal to the dissociation constant
The  the Km the  affinity for substrate
• Vmax
– Turnover number:
The number of substrates turned into products by a
single enzyme per unit of time (enzyme operating at
optimal velocity)
 3. Enzyme Inhibitors

a. Competitive inhibitors: are chemicals that resemble

an enzyme’s normal substrate and compete with it for
the active site.

Substrate
Enzyme

Competitive inhibitor
 Factors Influencing Enzyme Activity
• Competitive inhibition
 Competitive Inhibition

Competitive Inhibition
•As the amount of inhibitor increases,
Velocity vs [Substrate]
there is more competition at the active
site 1.60
• most pronounced at low levels 1.40
of S
1.20
•At higher [S], inhibition is “swamped [I] = x [I] = 2x

Initial velocity
[I] = 0
1.00
out”
0.80
•Vmax does not change
0.60
• processing rate not effected
•“Km” increases!!! 0.40

• the enzyme does not bind the 0.20

substrate as well in the presence 0.00

of inhibitor 0.00 0.50 1.00 1.50 2.00
substrate concentration [S]
 Competitive Inhibition
Competitive Inhibition
A Lineweaver-Burk plot allows the Lineweaver-Burk Inverse Plot
important parameters, Km and Vmax,
to be determined directly from an 8
equation rather than from an 7
extrapolation 6
[I] =2x

1/initial velocity
y = 0.6194x + 0.9995
5
[I] = x
1/Vmax = y intercept 4
y = 0.3983x + 0.9819
3
y = 0.2457x + 1.0103
Km/Vmax = slope of line 2

0
Get Vmax first, don’t forget inverse -5 0 5 [I] = 0 10 15
relationship!!! 1/substrate concentration [S]-1
Highest line most inhibited!!!!
 Competitive Inhibition
EI
Kic
Competitive S CI E S+E ES E+P
+
I
Vmax [S] Vmax [S]
v v
Km  [S] K m  [S] 5
5
No I

 [I] 
  1 
4 4
0.5V
 Kic  max

v, µmol/min
+CI
v, µmol/min

3 3
0.5Vmax

2 2 K
m
Kmapp
1 1
K
m

0 0
0 10 20 30 40 50 0 10 20 30 40 50
[S], mM [S], mM
 Competitive Inhibition
EI
Kic
Competitive S CI E S+E ES E+P
+
I
1 Km 1 1 1 K m 1 1
   
v Vmax [S] Vmax v Vmax [S] Vmax
2.5

2.5
2  [I]  Kmapp/Vmax
  1  +CI

 Kic  2
1/v, /µmol/min

1.5

1/v, /µmol/min
1.5
1 Km/Vmax
K /V
1/Vmax 1 m max
-1/K No I
m 0.5 1/Vmax
-1/Km app
-1/K m 0.5

0
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 0
1/[S], /mM -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
1/[S], /mM
 Enzyme Inhibitors
b. Noncompetitive inhibitors:
Inhibitors that do not enter the active site, but bind to
another part of the enzyme causing the enzyme to
change its shape, which in turn alters the active site.

Substrate Noncompetitive
Enzyme
Inhibitor
active site
altered
 Factors Influencing Enzyme Activity
• Noncompetitive inhibition
 Noncompetitive Inhibition

•Inhibitor and substrate DO NOT have common shape

•Binding is NOT at active site but causes a change in the substrate binding site

•Large amounts of inhibitor prevent access of substrate to binding site

•Large amounts of substrate CANNOT overcome inhibitory effect

 Noncompetitive Inhibition
Noncompetitive Inhibition
•As the amount of inhibitor Velocity vs [Substrate]
increases, the efficiency of
processing the product decreases 0.900
• same relative effect at all levels 0.800
of S 0.700 [I] = 0

Initial velocity
•Higher [S] cannot “swamp out” 0.600

effect; the “problem” is not at active 0.500

0.400
[I] = x
site
0.300
•Km does not change 0.200 [I] = 2x
• binding/dissociation not effected 0.100
•“Vmax ” decreases 0.000
•the enzyme is less efficient in 0.000 0.200 0.400 0.600 0.800 1.000 1.200
processing product substrate concentration [S]
 Noncompetitive Inhibition
A Lineweaver-Burk plot allows the Noncompetitive Inhibition
important parameters, Km and Vmax, Lineweaver-Burk Inverse Plot
to be determined directly from an
equation rather than from an
7
extrapolation y = 0.4504x + 1.7139
6

1/initial velocity
5 y = 0.307x + 1.2354
[I] = 2x
1/Vmax = y intercept 4

3 [I] = x
Km/Vmax = slope of line 2
y = 0.2461x + 1.0077

0
[I] = 0
Get Vmax first, don’t forget inverse -5 0 5 10 15
relationship!!!
1/substrate concentration [S]-1
Highest line most inhibited!!!
 Noncompetitive Inhibition
EI EI
S NCI E
Noncompetitive Kic Kiu
(mixed-type) S+E ES E+P
+ +
S E NCI
I I
Vmax [S] Vmax [S] 1 Km 1 1 1 K m 1 '
v v    
Km  [S] K m   ' [S] v Vmax [S] Vmax v Vmax [S] Vmax
5 5
 [I]  No I
  1 
4  Kic  4
0.5V
max

 [I] 
v, µmol/min

v, µmol/min
'  1 
3 0.5Vmax 3 + NCI

 Kiu 
2 2

Km
1 1
K
m

0 0
0 10 20 30 40 50 0 10 20 30 40 50
[S], mM [S], mM
 Noncompetitive Inhibition
EI EI
S NCI E
Noncompetitive Kic Kiu
(mixed-type) S+E ES E+P
+ +
S E NCI
I I
Vmax [S] Vmax [S] 1 Km 1 1 1 K m 1 '
v v    
Km  [S] K m   ' [S] v Vmax [S] Vmax v Vmax [S] Vmax
2.5 2.5
 [I] 
  1 
2  Kic  2 1/Vmaxapp
Km/Vmaxapp
 [I] 

1/v, /µmol/min
1/v, /µmol/min

'  1 
1.5 1.5
+ NC I
 Kiu 
1 Km/Vmax 1 K /V
m max
No I
1/Vmax -1/K
-1/Km m
0.5 0.5
1/Vmax
0 0
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
1/[S], /mM 1/[S], /mM
 Uncompetitive Inhibition

•Inhibitor and substrate DO NOT have common shape Binding is NOT at active site

•Binding of substrate causes a change in the shape of the enzyme that allows inhibitor
to bind

•Inhibitor binding prevents processing of substrate to product

•Larger amounts of substrate cannot overcome inhibitory effect

 Uncompetitive Inhibition
As the amount of inhibitor
increases:
• the binding is effected
• the efficiency of processing product
decreases
Km increases
•binding/dissociation effected
“Vmax ” decreases
•the enzyme is less efficient in
processing product
 Uncompetitive Inhibition
EI
Uncompetitive Kiu
(catalytic) S E UCI S+E ES E+P
+
Vmax [S] Vmax [S] I
v v 1 Km 1 1 1 Km 1 '
   
Km  [S] Km   ' [S] v Vmax [S] Vmax v Vmax [S] Vmax
5 5
No I

4 4
0.5Vmax

0.5Vmax
v, µmol/min

v, µmol/min
3 0.5Vmax  [I]  3 + UC I
'  1 
2
 Kiu  2 Kmapp
Km
1 1
K
m

0 0
0 10 20 30 40 50 0 10 20 30 40 50
[S], mM [S]. mM
 Uncompetitive Inhibition
EI
Uncompetitive Kiu
(catalytic) S E UCI S+E ES E+P
+
Vmax [S] Vmax [S] I
v v 1 Km 1 1 1 Km 1 '
   
Km  [S] Km   ' [S] v Vmax [S] Vmax v Vmax [S] Vmax
2.5 2.5

2 2 1/Vmaxapp

 [I] 
1/v, /µmol/min

1/v, /µmol/min
1.5 1.5 Kmapp/Vmaxapp
'  1 
 Kiu  K /V
m max
1 K /V 1 + UC I
m max
No I
1/V
-1/K max
m 0.5 -1/K 0.5
-1/Kmapp m
1/V
max
0 0
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
1/[S], /mM 1/[S]. /mM
 Where Effectors Bind

Effector

where does it bind?

At the At another site

Active Site

substrate competitive irreversible "designed "incidental site"

product inhibitor inhibitor site"

allosteric allosteric irreversible noncompetitive irreversible Uncompetitive

inhibitor activator inhibitor inhibitor inhibitor inhibitor
 Controls of Enzymatic Activity
• Inductive effects
– changing the amount of active enzyme present
– genetic control; irreversible covalent modification
• Regulatory effects
– changing the activity of enzymes already present
– LeChatelier control; allosteric control; reversible
covalent modification; hormonal control
 Inductive Effects

– Genetic control
• Turn genes on  make mRNA  make enzymes
– Irreversible covalent modification
• Proteolytic activation
• zymogen ----> active enzyme
• very common for digestive enzymes
 Regulatory effects
• LeChatelier
– every system responds to the levels of substrates and products
• Allosteric
– feedback: product of pathway providing information about the status of
metabolism
– forward activation: the product of an earlier reaction telling a later
enzyme to get ready
• Reversible covalent modification
– reversible phosphorylation usually on serine
– extra ramping up of enzymatic activity, sometimes related to hormonal
response; control points in metabolic pathways
• Hormonal control
– control from a distance (on organism level)
– binding to outside of cell OR entry into cell causes effect
 Factors Influencing Enzyme Activity
• Feedback inhibition