Enzymes
• Nearly all the reactions of the body are mediated by
enzymes
•
Enzymes are protein catalysts that increase the rate of
the reactions without being changed in the overall
process
• Catalysts for biological reactions
• 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
Nomenclature of enzymes
Enzyme has two names
a. Short Recommended name
b. Systematic name
Recommended name
•End in –ase,
•Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
•Describes function of enzyme
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
lactate dehydrogenase, adenylate cyclase…
•Common names of digestion enzymes still use which don't provide any
hint as pepsin and trypsin
Systematic name
The international union of Biochemistry and Molecular Biology (IUBMB)
developed a system for nomenclature in which enzymes are divided into
6 groups and sub classes. These names are unambiguous and
informative but sometimes long and difficult to be of general use.
•
Class
Reactions catalyzed
1.
Oxidoreductoases
oxidation-reduction
2.
Transferases
transfer group of atoms
3.
Hydrolases
hydrolysis
4.
Lyases
add/remove atoms to/from a double
bond
5.
Isomerases
6.
Ligases
rearrange atoms
combine molecules using ATP
Enzyme Action:
Lock and Key Model
•An enzyme binds a substrate in a region
called the active site
• Active site is a special pocket or cleft in
the enzyme molecule
• The active site contains amino acids
side chains that form a three
dimensional surface complementary to
the substrate
• Only certain substrates can fit the
active site
• The active site binds to the substrate
and form enzyme-substrate complex that
will dissociate into the enzyme and
product.
• Amino acid R groups in the active site
help substrate bind
Lock and Key
The active site of the unbound enzyme is complementary in shape to
that of the substrate
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
The enzyme changes
shape upon binding
substrate
The active site has a
shape complementary
to that of the
substrate only after
the binding
Enzyme-Substrate Complex
Cofactors
-Some enzymes associate with non-protein
cofactor that is needed for enzymatic activity
-These cofactor include metal ions (Zn, Fe) and
organic molecules called coenzymes that often
derivative of vitamins; NAD+, FAD, CoenzymeA..
-Holoenzyme refers to the enzyme with its
cofactor, Apoenzyme refers to the protein
portion of the holoenzyme and it dose not show
biological activity.
- Prosthetic group is a tightly bound enzyme
that dose not dissociate from the enzyme
Location of enzymes
Many enzymes are located into specific
organelles in the cell serve to isolate the
reaction substrate or product from each other
and to provide a special environment for a
reaction and to organize these reactions
Turnover number and catalytic efficiency
-Enzyme-catalyzed reactions are highly efficient, proceeding from 103 – 108 times
faster than unanalyzed reactions.
- The number of substrate molecules converted into product per enzyme per
second is called Turnover number, typically each enzyme molecule is capable of
transforming 100-1000 of substrate molecules into product per second
- Enzymes are highly specific, interacting with one or a few specific substrate
Enzymes could be enantiomers specific
How Enzymes work
The mechanism of action of enzymes can explained by two different modes
A. Energy changes during the enzyme-catalyzed reaction, enzyme provide
an energetically favorable reaction pathway different from unanalyzed
reaction
B. The active site chemically facilitates catalysis
Energy Changes during the reaction
- the reactant and the product are separated by energy gap or energy
barrier that called free energy of activation and it equals to the energy
difference between the energy of reactant and the energy of highenergy intermediate (Transition state)
A  T*  B
- The peak of energy represents the Transition state in which the highenergy intermediate is formed during the conversion of reactant into
product.
- because of large activation energy the rate of unanalyzed reaction is
slow
Rate of Reaction
-The substrate molecules should have sufficient energy to
overcome energy barrier to be converted into products.
- only few molecules have energy to pass the energy gap 
rate of reaction is determined by number of molecules that
is converted into the product.
- the lower the free energy of activation, the more molecules
have sufficient energy to pass over the transition state
the faster the rate of the reaction
The enzymes don’t change the free energies of the reactants and
products  don’t change the equilibrium of the reaction
Chemistry of the active site
The binding of a substrate to the binding site involves a complex molecular
machine chemical reactions  facilitate the conversion of substrate to product.
Many factors responsible for enzyme catalytic efficiency of enzymes:
-Transition state stabilization: the active site often act as a flexible molecular
template that binds substrate  stabilize the transition state  increase the
product production
- Active site provide catalytic groups that enhance the probability that transition
state forms, these groups participate in general acid base catalysis in which amino
acids can accept or provide protons, in other enzymes catalysis may involve the
transient formation of a covalent enzyme-substrate complex
Transition State
Factors Affecting Enzyme Action
Different enzymes show different response to changes in substrate
concentration, temperature, and pH
Reaction Rate (velocity of a reaction):
• Reaction rate is the number of substrate molecules converted to product per
unit time and is usually expressed as µmoles product formed per minute
• The rate of reaction (enzyme catalyzed) increases with substrate concentration
until a maximal velocity (Vmax), the plateau reflects the saturation with a
substrate of all available binding sites on the enzyme
Reaction Rate
 Increasing substrate
concentration increases the rate
of reaction (enzyme
concentration is constant)
 Maximum activity reached
when all of enzyme combines
with substrate
Maximum activity
substrate concentration
Maximum activity
Vmax
Reaction Rate
Most of enzymes show
hyperbolic dependence of
velocity on substrate
concentration
Vmax/2
Km
substrate concentration
Factors Affecting Enzyme Action: Temperature
Little activity at low temperature
•
Rate increases with temperature (the velocity increased with Tem
until a peak due to the increased number of molecules having
sufficient energy to pass over the energy barrier and form
product)
•
Most active at optimum temperatures (usually 37°C in humans)
•
Activity lost with denaturation at high temperatures decrease
Optimum temperature
the velocity
Reaction Rate
•
Low
Temperature
High
Factors Affecting Enzyme Action: pH
•pH affect the ionization of the amino acids in the active site.
•R groups of amino acids in the active site should have proper charge to bind with
the substrate the ionization or unionization is affected by the pH
•Maximum activity at optimum pH
•Tertiary structure of enzyme is correct
•Narrow range of activity,
and the pH optimum varies
for different enzymes
Reaction Rate
•Most lose activity in low or high pH, extremes of pH can also lead to
denaturation of the enzyme because the structure of active protein depends on
the ionic character of the amino acid
Optimum pH
3
5
11
pH
7
9
Each enzyme has
its optimum pH
Michaelis-Menten Model
Michalis and Menten proposed model that accounts for most features of enzymecatalyzed reactions. In this model the enzyme reversibly binds its substrate to
form ES complex that subsequently breaks down to product
E+S
k1
k2
ES
E+P
Where
k-1
E: enzyme
S: substrate
ES: enzyme-substrate complex
P: product
k1 k-1 k2 are rate constants
Michaelis-Menten Equation
The Michaelis-Menten Equation describes how reaction velocity varies with
substrate concentration
V [S]
Rate v
=
km + [S]
Assumptions Required for Michaelis-Menten Equation
• E and S combine to form ES complex
• There is only a single substrate, the concentration of S is much greater than
concentration of enzyme the amount of the substrate bound by the enzyme at
any one time is small.
• E + S = ES rapidly reaches equilibrium: steady state assumption, concentration
of ES is constant, dose not change with time the rate of formation of ES is
equal to that of the breakdown of ES (to E and P)
•Reaction from P is irreversible
• only the initial reaction velocities are used in the analysis of enzyme reactionsthe rate of reaction at zero time, at that time the concentration of eth product
is very small  the rate of the back reaction from P to S can be ignored
Michaelis-Menten Equation
Vo: initial reaction velocity
Vmax: maimal velocity
Km: michaelis-menten constant=
(k-1+K2)/K1
[S]: concentration of substrate
V max[S]
vo
=
km + [S]
Michaelis-Menten Model
Michaelis-Menten Model
Conclusions about Michaelis-Menten kinetics
Characteristics of Km:
 The Km constant is a characteristics
of an enzyme and particular substrate
 Km reflects the affinity of the
enzyme for a particular substrate
 Km numerically equal to the
concentration substrate at which the
reaction velocity is equal to ½ Vmax.
 Km dose not vary with concentration
of the enzyme
 low Km  high affinity; low [S] is
needed to half-saturate the enzyme
 large Km  low affinity; high [S] is
needed to half-saturate the enzyme
Relationship between the velocity to
enzyme concentration: the rate of
reaction is directly proportional to the
enzyme concentration at all substrate
conc. if the enzyme concentration is
halved  the Vo is reduced to half
Order of reaction:
When [S] << Km, V (velocity of reaction)
is roughly proportional to [S] the rate
of the reaction is first order reaction
When [S] >> Km, the V is constant and
equal Vmax. The rate of reaction is
independent on the [S] the reaction
rate is Zero order
Double reciprocal of Michaelis-Menten equation
Lineweaver-Burk Plot
When V is plotted versus the [S], it is not always possible to determine
the Vmax or Km from the graph because of gradual upward slope of the
hyperbolic curve
vo =Vmax [S] / (Km + [S])
(Michaelis-Menten Equation)
Equation of straight line: y= ax + b (a = slope, b = y intercept
Linear Transform: take reciprocal of each side of equation
1/vo = Km/Vmax • 1/[S] + 1/V
y
=
a
x
+
b
Double reciprocal of Michaelis-Menten equation
Lineweaver-Burk Plot
Lineweaver-Burk Double Reciprocal Plot
1/vo = Km/Vmax • 1/[S] + 1/Vmax
Lineweaver-Burk line allow
us to determine the Vmax
and Km simply
The x intercept is -1/km
The y intercept is 1/Vmax
What do we mean by inhibitors?
Enzyme Inhibition
• Enzyme inhibitor: any substance that can reduce the velocity of an
enzyme-catalyzed reaction and cause a loss of catalytic activity
•
Inhibitors can be reversible or irreversible.
- Reversible inhibitors bind to enzymes through a non-covalent bonds 
Upon dilution the EI (enzyme-inhibitor) complex dissociate and recover
the enzyme activity, may be competitive or noncompetitive
- Irreversible inhibition occur when the inhibited enzyme can not
recover its activity by dilution. The inhibitors form a covalent bonds
with the active site enzyme or destruction of the protein structure of
the enzyme
Irreversible Inhibitors
Competitive inhibitors
This type of inhibition occurs when the inhibitors bind reversibly to the
same site that the substrate normally occupy  compete the substrate for
that site
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
Competitive inhibitors
Vmax: the effect of the competitive inhibitors is reversed by increasing the [S].
At sufficient high substrate concentration, the reaction velocity reaches the Vmax
observed in the absence of the inhibitors. The y-intersect is unchanged
Km: a competitive inhibitors increases the apparent Km for a given substrate in
the presence of this inhibitors , more substrate is needed reach the Vmax. The x
intersect is changed indicating that the apparent Km is increased
Non-Competitive Inhibitors
Inhibitor binds at a site distinct from the substrate
site
It may bind to free E or to ES.
Once bound it will prevent P formation.
And the affinity of the I to both E and Es is the same.
Noncompetitive Inhibition
• Non competitive inhibition occurs when the inhibitor at a site
distinct from the substrate site
• A noncompetitive inhibitor does not have a structure like
substrate.
• Alter the shape of enzyme and active site Substrate cannot
fit altered active site
• It binds either to the enzyme or to the ES complex No
reaction occurs
• Effect is not reversed by adding substrate
• It may bind to free E or to ES. Once bound it will prevent P
formation.
• And the affinity of the I to both E and ES is the same.
• Non-competitive inhibitors decrease the Vmax, and can not be
overcome by increasing the substrate concentration
• Non-competitive inhibitors don’t affect the Km, the enzyme
show the same Km in the absence or the presence of the
inhibitors
Enzyme inhibitors could be used as drugs
Different types of enzyme inhibitors
I2
I1
NONCOMP
COMP
I0
Allosteric Enzymes and Allosteric Regulation
Allosteric enzymes: are regulated by molecules
called effectors (modulators) that bind noncovalently at site other than the active site.
- Allosteric enzymes show Sigmoidal curve and
don't follow Michalaelis-menten rules
- Allosteric modulators affect the enzyme
affinity to its substrate and /or modify the
maximal catalytic activity of the enzyme
- Enzyme regulation is essential to coordinate
the numerous metabolic processes
-Most of enzymes respond to changes of
substrate concentration  an increase in the
substrate conc. lead to increase in the rate of
enzyme action  return the substrate conc. to
normal level
-Some enzymes respond to allosteric
effectors or covalent modification that
affect the velocity of enzyme
Allosteric regulators (effectors)
-Effectors that inhibit enzyme activity are termed negative effectors
while those increase the enzyme activity are called positive effectors
- Homotropic effectors:
•The substrate itself acts as effector, usually allosteric substrate are
positive effector.
•The presence of substrate molecule at allosteric site will increase the
catalytic activity of the substrate-binding site  sites cooperativity 
Sigmoidal curve of V vs [S] not hyperbolic
-Heterotropic effectors
The effectors are different from substrate
Feed back inhibition
A B C DE
This enzyme has allosteric site that can bind to E
The final product may have a feedback inhibition of on the enzyme that
convert AB
Feed back inhibition serves to coordinate the flow of substrate
molecules through a series of reactions with the need of the cell.
Enzymatic Regulation
Covalent modification-Protein phosphorylation
- Many enzymes may be
regulated by covalent
modification; addition or
removal of phosphate
group
-Phsphorylation occurs at
the –OH of serine,
threonine or tyrosine
residues
- Phosphorylation process
could be Activation or
Deactivation process 
depending on the enzyme
itself
active
Inactive or
Induction and repression of enzyme synthesis
•Cells can regulate the amount of eth enzyme present, usually by
altering the rate enzyme synthesis
•The induction or repression synthesis of protein leads to an alteration
of the total number of enzyme population  increasing the number of
active sites and Not affecting the enzyme activity.
The End
Learning Objectives
•
•
•
•
•
Gibbs Free Energy Diagram –
illustrates activation energy and two
chemical steps
Define Enzyme – protein, catalyst,
specific, activates substrate
Binding of Substrate to Active Center
Transition State binds tighter than
substrate or product
Mechanism of Chymotrypsin – DHS
Learning Objectives
1. Lineweaver-Burk plot
2. Competitive, Uncompetitive and
Noncompetitive Inhibition
3. Possible models of inhibition
4. Suicide and irreversible
inhibition
5. Natural inhibitors
Competitive inhibitors
Uncompetitive Inhibitors
Inhibitor binds at an
allosteric site, but only to
the ES complex
The slopes of 1/Vo vs.
1/[S] are unchanged but
Vmax is lower, and so is
the apparent [S] needed to
reach 1/2 Vmax = Km.
Vo
Enzyme inhibitors could be used as drugs
Different types of enzyme inhibitors
COMP
UNCOMP
NONCOMP