BIO2102 - Laboratory | Biochem | Midterms

Chapter 1: Enzymes: Principles, ● Substrate-binding and

Classification & Mechanism of Action Catalytic site of the enzyme.
● Complementary to the
shape of the substrate
Chapter Outline
I. Introduction to Enzymes
Substrate (in yellow)
A. What are Enzymes?
B. Enzyme Structure ● Molecules which are acted upon by
II. Classification of Enzymes enzymes
III. Enzyme Activity and Kinetics ● in the case of Amylase, the substrate
IV. Mechanisms of Enzyme Action shown is a short chain polysaccharide
A. The and Lock-and-Key Model which will be broken down into smaller
B. The Induced Fit Model sugar units.
V. Enzyme Regulation and Inhibition
Allosteric Site
● Location in an enzyme for molecules
Introduction to Enzymes to either activate or inhibit enzyme
Cofactors
What are Enzymes? ● Inorganic ions needed by
some enzymes to be active.
● Amylase needs Chloride
● Proteins that act as biological catalysts (in dark green) and Calcium
● In biological conditions, biomolecules (in light green) to function.
are stable. Coenzymes
● For them to be metabolized, most ● Cofactors which are organic molecules
reactions in the body rely on the action ● Coenzyme e.g: B vitamins
of enzymes to proceed.
Note: Some enzymes need a coenzyme to function.
The inactive protein part of the enzyme is known as an
Apoenzyme.

Once a coenzyme is bound and the enzyme becomes

active, it is known as a Holoenzyme.

Classification of Enzymes
Enzymes are classified into six (6) major
groups based on the reaction they catalyze.

1. Oxidoreductases
● Catalyzes oxidation and reduction
reaction.
● Requires a coenzyme to function (in
Enzyme Structure the form of NADP+ or NAD+)

Amylase - an enzyme found in saliva and

pancreas
● Responsible for hydrolysis of sugars
during digestion

Active site (in red) 2. Transferases

BIO2102 - Laboratory | Biochem | Midterms
● catalyzes the transfer of a functional
group from one molecule to another.

Enzyme Activity and Kinetics

- Enzyme activity is the rate at which an
enzyme converts substrate into
product.
- The following can affect enzyme
activity:
1. Substrate Concentration
2. Enzyme Concentration
3. Hydrolases 3. Temperature
● Catalyze the removal of bonds via 4. pH
hydrolysis - To fully understand the mechanism of how
these factors affect enzyme activity, we first
look at the basics of enzyme kinetics.

The Basics of Enzyme Kinetics

● Enzyme Kinetics is used to determine
4. Lyases
the rate of the reaction and how it
● Catalyze the addition of groups to
changes in response to changes in
double bonds
concentration, temperature, and pH.
● Can also catalyze the formation of
double bonds by removal of groups
The Effect of Substrate Concentration
● The concentration of the Substrate
[S] is a key factor affecting the rate of
a catalyzed reaction.
● Assuming that the concentration of the
Enzyme, [E], is held constant, we can
model a step-wise reaction for the
enzyme and substrate.
5. Isomerases
● Catalyze the interconversion of a
molecule to its isomers

STEP 1. The enzyme [E] firsts binds to the

6. Ligases
substrate [S] to form the Enzyme-
● Catalyze the formation of bonds via
condensation reactions. (uses ATP) Substrate Complex [ES] in a fast,
reversible step.
BIO2102 - Laboratory | Biochem | Midterms

The Effect of Temperature

STEP 2. The [ES] then breaks down to

yield the free enzyme, [E] and the reaction
product, [P]. This is the ratelimiting step
of the overall reaction.

● The rate of enzyme-catalyzed

reactions generally increase with
increasing temperature
● The rate usually doubles every 10℃
increase in temperature.
From the graph, we can infer the following: ● However, at temperatures above 50℃,
enzyme activity will generally decline
due to degradation of protein
● During STEP 1, at low [S], the rate,
structure.
V, is directly proportional to [S] .
The Effect of pH
Why?
● During STEP 2, [E] becomes
infinitely small, because all enzymes
are now used as the ES complex.
● Since the enzyme is now fully
saturated, ↑[S] will have no effect
on V, hence the plateau observed.
The Effect of Enzyme Concentration
● When the concentration of the
● The rate of enzyme-catalyzed
enzyme is significantly lower than
reactions are highly pH dependent.
the concentration of the substrate, ● The optimum pH range of an enzyme
the rate of an enzyme-catalyzed depends on the ionizable side chains
reaction is directly dependent on the present in the amino acid of the active
enzyme concentration. site.
● The reaction rate increases as the
concentration of the enzyme is
increased.
BIO2102 - Laboratory | Biochem | Midterms
Mechanisms of Enzyme Action Enzyme Regulation and Inhibition
Enzyme Regulation
The Lock-and-Key Model ● refers to the control of enzyme activity
through modification of the enzyme.
● Enzymes may need to be regulated to
keep concentrations of intermediate
metabolites relatively constant.
● The following are the different
mechanisms used in enzyme
regulation:
○ Feedback Control
○ Proenzymes
Assumptions: ○ Allosteric Modulation
● The enzyme is a rigid, three- ○ Covalent Modification
dimensional structure with a highly ○ Isoenzymes
restricted active site.
Feedback Control
● A high degree of similarity between the
● The formation of the product inhibits
shape of the substrate and the
an earlier reaction in the metabolic
geometry of the binding site.
sequence.
● The shape of the substrate is
● Case: The synthesis of the
complementary to the active site,
isoleucine in [Link]. The pathway of
similar to how a key fits perfectly in
the synthesis of the amino acid
a lock.
isoleucine has five steps, each
catalyzed by a different enzyme. If a
The Induced Fit Model cell begins to produce more isoleucine
than needed, the high concentration of
isoleucine inhibits the enzyme activity
of enzyme 1.

Assumptions:
● Proteins have three-dimensional Proenzymes
flexibility which allows the enzyme to ● also called zymogens or proproteins,
change the conformation of the these are inactive precursors of
active site to accommodate the shape enzymes that become active when a
of the substrate. specific peptide bond is cleaved.
● Allows the explanation of the nature of ● Case: The activation of the enzyme,
the transition state, and the lower Trypsin. Trypsin is an enzyme used for
activation energy of the reaction. protein digestion which the body could
not synthesize directly. Trypsinogen,
the proenzyme of Trypsin, is secreted
by the pancreas during digestion. Once
BIO2102 - Laboratory | Biochem | Midterms
it enters the duodenum, the peptide ● change in the protein primary structure
bond between Lys15 and Ile16 is through the covalent addition of a
cleaved to form Trypsin. functional group to specific amino acid
residues in the apoenzyme.
● The most common type is
Phosphorylation.

Phosphorylation (Tyr, Ser, Thr, His)

Allosteric Modulation
● occurs when a molecule known as a
regulator or effector, binds to a site Adenylylation (Tyr)
other than the active site – known as
the regulatory site – and alters the
shape of the active site.
● the product of this modification is
known as an allosteric enzyme.

Isoenzymes
● also called isozymes, are different
quaternary structures of the same
enzyme.
● although isoenzymes are structurally
● Allosteric enzymes can be equivalent, each form may have
described in two forms. The R form varying amino acid sequences,
(in red, stands for “Relaxed”) is enzymatic activity, and may occur in
different subcellular distributions
more likely to bind to the substrate,
(either soluble or membrane
than the T form (in orange, stands
bound).
for “Taut”). The enzyme is in ● Case: The Five Isoenzymes of
constant equilibrium between the Lactate Dehydrogenase and their
two forms. General Distribution among
Tissues.
Enzyme Inhibition
● refers to the action of molecular agents
that interfere – either by slowing or
stopping – enzyme activity.
● Some of the most important
pharmaceutical agents are enzyme
inhibitors.
Covalent Modification ● Can either be Reversible or
Irreversible Inhibition.
BIO2102 - Laboratory | Biochem | Midterms
Reversible Inhibition
● the inactivation of the enzyme
through noncovalent
association/dissociation reactions.
● Can be competitive,
noncompetitive, and
uncompetitive inhibition
Competitive Inhibition Effect on the Michaelis-Menten Equation:
● In competitive inhibition, the inhibitor 𝑉 𝑚𝑎𝑥 = Lowered
binds directly to the active site of the
𝐾𝑚 = Lowered
enzyme preventing the binding of the
Irreversible Inhibition
substrate.
● binds covalently with or destroys an
● Competitive inhibitors resemble the
essential functional group of the
substrate.
enzyme.
● Completely alters or inactivates the
enzyme.

Effect on the Michaelis-Menten Equation:

𝑉 𝑚𝑎𝑥 = No Effect
𝐾𝑚 = Increases
Non-Competitive Inhibition
● In non-competitive inhibition, the
inhibitor binds to a site other than
the active site and can bind to either
the enzyme or the substrate.
● Non-competitive inhibitors do not
resemble the shape of the substrate.

Effect on the Michaelis-Menten Equation:

𝑉 𝑚𝑎𝑥 = Decreases
𝐾𝑚 = No Effect
Uncompetitive Inhibition
● In uncompetitive inhibition, the
inhibitor binds to a site other than
the active site but only in the
Enzyme-Substrate complex