Lecture 4 (Part 2) - Enzymes and its types

LECTURE 4 – PART 2
Course: BIT-103 (Biochemistry I)
BS – Biotechnology (2nd
semester)
ENZYMES
Enzyme kinetics
Chapter 6 – Enzymes
Lehninger Principals of biochemistry 6th Edition
BY
NAFEESA QUDSIA HANIF, PhD

Enzyme Kinetics
• Kinetics is the study of the rate at which
compounds react
• Rate of enzymatic reaction is affected by
– Enzyme
– Substrate
– Effectors
– Temperature

Michaelis–Menten equation
The Michaelis–Menten equation is the rate equation
for a one-substrate enzyme-catalyzed reaction. This
equation relates the
• initial reaction rate (v0),
• the maximum reaction rate (Vmax),
• the initial substrate concentration [S]
• Michaelis constant KM—a measure of the substrate-
binding affinity.

Michaelis–Menten equation
Let’s consider a reaction in which a substrate (S)
binds reversibly to an enzyme (E) to form an
enzyme-substrate complex (ES), which then
reacts irreversibly to form a product (P) and
release the enzyme again.
S + E ES → P + E
⇌

Two important terms within Michaelis-Menten kinetics
are:
Vmax – the maximum rate of the reaction, when all
the enzyme’s active sites are saturated with substrate.
Km (also known as the Michaelis constant) – the
substrate concentration at which the reaction rate is 50%
of the Vmax. Km is a measure of the affinity an enzyme has
for its substrate, as the lower the value of Km, the more
efficient the enzyme is at carrying out its function at a
lower substrate concentration.
The Michaelis-Menten equation for the reaction above is:

Why KM is 50% of Vmax
KM is a constant and is not Vmax/2 as they have different
dimensions.
But by the Michaelis Menten equation,
V=Vmax [S]/KM+[S]
So, when
[S]= KM, we have
V=Vmax[S]/[S]+[S]
V = Vmax/2.
Hence, KM is the substrate concentration, at which we
would have
V=Vmax/2

This equation describes how the initial rate of reaction
(V) is affected by the initial substrate concentration ([S]).
It assumes that the reaction is in the steady state, where
the ES concentration remains constant.
When a graph of substrate concentration against the rate
of the reaction is plotted, we can see how the rate of
reaction initially increases rapidly in a linear fashion as
substrate concentration increases (1st order kinetics).
The rate then plateaus, and increasing the substrate
concentration has no effect on the reaction velocity, as all
enzyme active sites are already saturated with the
substrate (0 order kinetics).

Fig 1 – Graph of the rate of reaction against substrate concentration,
demonstrating Michaelis–Menten kinetics, with Vmax and Km highlighted

This plot of the rate of reaction against substrate
concentration has the shape of a rectangular
hyperbola. However, a more useful
representation of Michaelis–Menten kinetics is a
graph called a Lineweaver–Burk plot, which
plots the inverse of the reaction rate (1/r) against
the inverse of the substrate concentration (1/[S]).

Lineweaver–Burk plot – Double Reciprocal???
The Lineweaver Burk plot is
a graphical representation
of enzyme kinetics. The x-
axis is the reciprocal of the
substrate concentration, or
1 / [S], and the y-axis is the
reciprocal of the reaction
velocity, or 1 / V. In this
way, the Lineweaver Burk
plot is often also called a
double reciprocal plot.

Fig 2 – Different types of enzyme inhibition as shown on a
Lineweaver-Burk plot

Eﬀect of [S] on reaction rate
• The relationship between [S] and V0 is similar for
most enzymes (rectangular hyperbola)
•Mathematically expressed by Michaelis-
‐
Menten equation:
Km [S ]
• Deviations due to:
– limitation of measurements
– substrate inhibition
– substrate prep contains inhibitors
– enzyme prep contains inhibitors
V 0 
Vmax
[S ]

Eﬀect of Substrate Concentration
V0 approaches but never reaches Vmax
(Michaelis constant)
[S] >> [E]

Determination of Kinetic Parameters
Nonlinear Michaelis-Menten
‐ plot should be used to
calculate parameters Km and Vmax.
Linearized double-reciprocal
‐ (Lineweaver-Burk)
‐ plot
is good for analysis of two-substrate
‐ data or
inhibition.

Lineweaver-Burk
‐ Plot:
Linearized, Double-Reciprocal
‐
V 0 V max[S]
1 Km [S]


Two-Substrate
‐ Reactions
• The rate of a bisubstrate reaction can also be analyzed
by Michaelis-Menten
‐ kinetics. Enzymes catalyzing
polysubstrate reac0ons have Km for each of their
substrates
• Kinetic mechanism: the order of binding of
substrates and release of products
• When two or more reactants are involved, enzyme
kine0cs allows to dis0nguish between diﬀerent kine0c
mechanisms
– Sequential mechanism (involving a ternary
complex)
– Ping-Pong
‐ (double displacement) mechanism

Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity
• Irreversible inhibitors (inactivators) react with the enzyme
• One inhibitor molecule can permanently shut oﬀ one enzyme molecule
• They are often powerful toxins but also may be used as drugs
• Reversible inhibitors bind to and can dissociate from the enzyme
• They are often structural analogs of substrates or products
• They are often used as drugs to slow down a speciﬁc enzyme
• Reversible inhibitor can bind:
• to the free enzyme and prevent the binding of the substrate
• to the enzyme-substrate
‐ complex and prevent the reac0on

• Competes with substrate for binding
– Binds active site
– Does not aﬀect catalysis
– many competitive inhibitors are similar in
structure to the substrate, and combine with the
enzyme to form an EI complex
• No change in Vmax; apparent increase in Km
• Lineweaver-Burk:
‐ lines intersect at the y-axis
‐
at –1/Vmax
Competitive Inhibition

Competitive Inhibi@on
FIGURE 6–15 Types of reversible inhibitions.
(a) Competitive inhibitors bind to the enzyme’s active site; KI is the
equilibrium constant for inhibitor binding to E

Uncompetitive Inhibition
• Only binds to ES complex
• Does not aﬀect substrate binding
• Inhibits catalytic function

Uncompetitive Inhibition
FIGURE 6–15 (b) Uncompetitive
inhibitors bind at a separate site,
but bind only to the ES complex;
K’I is the equilibrium constant for
inhibitor binding to ES.

Mixed Inhibition
• Binds enzyme with or without substrate
― Binds to regulatory site
― Inhibits both substrate binding and catalysis

Mixed Inhibition
FIGURE 6–15 (c) Mixed inhibitors
bind at a separate site, but may
bind to either E or ES.

Enzyme activity depends on pH
• Enzymes have optimum pH ranges at which
their activity is maximal:
– Activity decreases at higher or lower pH values
– Due to the physical and chemical properties of
amino acids and their side
chains

Regulatory Enzymes
•Each cellular metabolism pathway has one or
more regulatory enzymes (enzymes that have a
greater effect on the rate of the overall sequence)
•They show increased or decreased activities in
response to certain signals (function as switches)
•Generally, the first enzyme in a pathway is
a regulatory enzyme (not always true!)

Regulatory Enzymes
• Classes of regulatory enzymes:
allosteric enzymes (affected by
reversible noncovalent binding of allosteric
modulators)
nonallosteric/covalent enzymes (affected
by reversible covalent modification)
 regulatory protein binding enzymes
(stimulated or inhibited by the binding of separate
regulatory proteins)
 proteolytically activated enzymes (activated
by the removal of some segments of their
polypeptide sequence by proteolytic cleavage)

Allosteric Enzymes
•Allosteric enzymes function through
reversible, noncovalent binding of regulatory
compounds (allosteric modulators, aka allosteric
effectors)
• Modulators can be stimulatory or inhibitory
•Sometimes, the regulatory site and the
catalytic site are in different subunits
• Recall: homotropic (The modulator is also the substrate for
the enzyme. A well-known example is O2, which is a homotropic
allosteric modulator of hemoglobin) and heterotropic (The
modulator and the substrate are different molecules) enzymes
•Conformational change from an inactive T

FIGURE 6–31 Subunit interactions in
an allosteric enzyme, and interactions
with inhibitors and activators. In many
allosteric enzymes the substrate-
binding site and the modulator-binding
site(s) are on different subunits, the
catalytic (C) and regulatory (R)
subunits, respectively. Binding of the
positive (stimulatory) modulator (M) to
its specific site on the regulatory
subunit is communicated to the
catalytic subunit through a
conformational change. This change
renders the catalytic subunit active
and capable of binding the substrate
(S) with higher affinity. On dissociation
of the modulator from the regulatory
subunit, the enzyme reverts to its
inactive or less active form.

Allosteric Enzymes
•Allosteric enzymes are generally larger and
more complex than nonallosteric enzymes with
more subunits
•Aspartate transcarbamoylase (ATCase) catalyzes
an early step in pyrimidine nucleotide biosynthesis
•Allosteric enzyme, composed of 6 catalytic
subunits (organized as 2 trimeric complexes) and 6
regulatory subunits (organized as 3 dimeric
complexes)
• Catalytic subunits function cooperatively
•Regulatory subunits have binding sites for ATP

Regulated Steps Are Catalyzed by
Allosteric Enzymes
•Feedback inhibition – regulatory enzymes
are specifically inhibited by the end product of
the pathway whenever the concentration of the
end product exceeds the cell’s requirements
• Heterotropic allosteric inhibition

Threonine dehydratase (E1) is
specifically inhibited allosterically
by L-isoleucine, the end product of
the sequence, but not by any of the
four intermediates (A to D).
Ile does not binds to the active site
but to a regulatory site on the
enzyme.
The binding is reversible: if
[Ile]↓ rate of Thr dehydration ↑

Lecture 4 (Part 2) - Enzymes and its types

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