enzyme kinets slides

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BIOC708
ADVANCED BIOCHEMICAL CORE TOPICS
MODULE AIM:
 Introduce students to advanced application of gene products in biotechnology
 Lecturer and Module coordinator– Dr Karen Pillay
ASSESSMENT:
 Test and Assignments (25%)
 Exam (75%)
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BIOC708
Application of enzymes in Medicine and Industry
1. What are enzymes?
2. Fundamentals of enzyme kinetics
i. Enzyme nomenclature
ii. Mechanism of enzyme action
iii. Effect of pH and temperature on enzyme activity
iv. Determination of Vmax and Km
v. Enzyme inhibition
3. Sources of enzymes
4. Industrial use of enzymes
5. Preparation and Purification of enzymes
6. Immobilization of enzymes
7. Analyses of scientific publications

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Enzymes
The Nature of Enzymes
The Enzyme-Substrate Complex
Regulation of Enzymes
Enzymes in Medicine

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What Enzymes Are IS
What They Do
 Biological catalysts - contain very large proteins
• Are highly efficient catalysts
 Permit reactions to ‘go’ at conditions that the body
can tolerate.
 Each enzyme molecule can process thousands or
even millions of reactant molecules every minute
(turnover rate).
• Are very specific - catalyze the reaction of one or only a
few types of molecules (substrates).
• Regulate which cellular reactions take place (out of
thousands of possible reactions that might otherwise
occur in the cell)
• How does regulation occur?

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Some Properties of Enzymes
 Specificity - The limitation of
enzyme action to just one
substrate or one type of bond.
 Turnover rate - How many
molecules can be helped to react
by each molecule of enzyme per
second.
 Action low except at pH and
temperature in vivo.

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A + B [A------B] C
Reactants Transition State Product
The transition state theory indicates that products are only formed
after the reactant species has;
a) Collided in an optimum spatial orientation that will progress to
a chemical reaction between them.
b) Acquired sufficient energy to reach a transition state wherein
the bond breaking or bond forming chemistry is at some stage
of development.

7. Figure 1: A catalyst reduces the free energy of activation ∆G‡
and not the standard free energy ∆G° of the reaction.
The action of a biological catalyst
enhances the rate of a chemical
reaction because they decrease the
free energy of activation (∆G‡)
required for the reaction.
That is a catalyst provides an
alternative reaction pathway that
requires less energy. Therefore the
transition state can be attained with
greater efficiency and frequency.

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Enzyme Nomenclature
 Naming is pretty easy compared to other
compounds.
 Name is based on:
 - what it reacts with
 - how it reacts
 - add -ase ending
 Examples
 lactase - enzyme that reacts with lactose.
 pyruvate decarboxylase - removes carboxyl
from pyruvate.

9. Six Major Classes of enzymes
CLASS 1: OXIDOREDUCTASES
• Oxidoreductases catalyze oxidation reduction reactions.
• Most of these enzymes are referred to as dehydrogenases,
but some are called oxidases, peroxidases, oxygenases or
reductases

10. CLASS 2: TRANSFERASES
• Transferases catalyze group-transfer reactions and many of
them require the presence of a coenzyme. In these reactions,
a portion of the substrate molecule usually binds covalently
to the enzyme or coenzyme. This group includes the kinases.

11. CLASS 3: HYDROLASES
• Hydrolyases catalyze hydrolytic reactions. They are a special
class of transferases, with water serving as the acceptor of
the group transferred.

12. CLASS 4: LYASES
• Lyases catalyze nonhydrolytic and nonoxidative elimination
reactions, or lysis of substrate reactions, generating a
double bond. In the reverse reaction, lyases catalyze the
addition of one substrate to a double bond of a second
substrate. A lyase that catalyses an addition reaction in cells
is often termed a synthase.

13. CLASS 5: ISOMERASES
• Isomerases catalyse intramolecular rearrangement
reactions, i.e. isomerization reactions. They are among the
simplest of reactions due to the fact there is only one
substrate and a single product.

14. CLASS 6: LIGASES
• Ligases catalyse ligation or joining of two substrates. These
reactions are endothermic in that they require an input of
chemical potential energy of a nucleoside triphosphate such
as ATP. Ligases are referred to as synthetases.

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Binding Model
 Enzyme molecules usually have two components:
a polypeptide portion, called an apoenzyme, and a
nonpolypeptide portion, called a
cofactor. If the cofactor is organic, it is
called a coenzyme. The active site is on
the apoenzyme.
In (a), the enzyme
and substrate form
a complex. In (b),
the reaction oc-
curs. In (c), the
products separate
from the enzyme.

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The Active Site
 Enzymes are typically HUGE proteins, yet
only a small part is actually involved in a
reaction.
The active site has two
basic components.
catalytic site
binding site
Model of
triose-p-isomerase

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Characteristics of
Enzyme Active Sites
 Catalytic site - this is where the reaction actually occurs.
 Binding site - this is the area that holds the substrate in
proper place.
 Enzymes uses weak, non-covalent interactions to hold the
substrate in place based on R groups of amino acids.
 Shape is complementary to the substrate and determines
the specificity of the enzyme (“lock and key” or “induced fit”
models).
 Sites are pockets or clefts on the enzyme surface.

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Introducing E and S,
the Players in an Enzymatic Reaction
Catalytic site
Binding
site
Substrate
(S)
Enzyme (E)

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Step 1 in Enzyme-catalyzed
Reaction
 The first step is combination of E and S to
form the enzyme-substrate complex
. E + S ES
. Enzyme Substrate Complex
+

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Step 2
 In the second step the complex goes
through a transition state. An intermediate
species is then formed.
. ES ES*
note
change

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Step 3
 The reaction changes ES* to EP. The
activated enzyme-substrate complex then
becomes an enzyme-product complex.
. ES* EP

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Step 4
 The enzyme and product separate. Now
the product is finally made and the
enzyme is ready for another substrate.
. EP E + P

23. Effect of pH on Enzymatic Activity
• The pH activity profile of any enzyme is dependant on the acid-
base behaviour of the enzyme and the substrate,
• i.e. any ionizable R-group belonging to the active site (catalytic
AA residue);
• ionizable R-groups of the structural AA residues and
• ionizable groups belonging to the substrate molecule that is
involved in binding interactions with the enzyme.
• It is notable that the optimal pH of an enzyme is not necessarily
the pH of the cellular environment. Thus it may be that the
intracellular pH may exert some control on the activity of an
enzyme.

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25. Effect of pH on Enzymatic Activity
• Most enzymes have an optimal pH at which their activity is
maximal.
• If an enzyme is kept saturated with substrate at all pH values
tested, many enzymes would have a pH activity profile that is
characterized by a "bell-shaped" curve.
• However the pH activity profiles of certain enzymes may vary
considerably.

26. Effect of Temperature on Enzymatic Activity
• The rates of enzyme catalysed reactions generally increase with
temperature, within the T range in which the enzyme is stable
and retains full activity.
• Although enzyme catalyzed reactions have an optimum
temperature, the peak in such a plot of catalytic activity versus
temperature results because enzymes being proteins are
denatured by heat and become inactive.
• Most enzymes are denatured or inactivated at temperatures
above 55C to 60C.

27. KINETIC CONSTANTS INVOLVED IN ENZYME ACTIVITY AND CATALYTIC PROFICIENCY

28. KINETIC CONSTANTS INVOLVED IN ENZYME ACTIVITY AND CATALYTIC PROFICIENCY
Figure 2
Meanings of kcat and kcat/Km. The catalytic constant (kcat) is the rate constant for conversion of the
ES complex to E + P. It is measured most easily when the enzyme is saturated with substrate (region
A on the Michaelis–Menten curve shown). The ratio kcat/Km is the rate constant for the conversion
of E + S to E + P at very low concentrations of substrate (region B). The reactions measured by these
rate constants are summarized below the graph.

29. MEASUREMENT OF Km and Vmax
Figure 3
Double-reciprocal (Lineweaver-Burk) plot. This plot is derived from a linear transformation of the
Michaelis-Menten equation. Values of 1/ v0 are plotted as a function of 1/[S] values.

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Regulation of Enzymes
 Enzymes can be switched on and off by initiators,
effectors, inhibitors, genes, poisons, hormones,
and neurotransmitters.
 In the cases above, one active site is being acti-
vated by an event occurring elsewhere on the
enzyme, an allosteric effect.

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Enzyme Inhibition
 Allosteric effects can be inhibiting as well as activating, as in (a) above.
 (b) illustrates so-called competitive inhibition, in which a non-substrate
molecule with a shape similar to that of the true substrate can compete with
the substrate for attachment to the active site.

32. Competitive inhibition
•A competitive inhibitor (I) is a substance that reversibly
binds with the free form of an enzyme (E) to produce a
binary EI complex that is incapable of binding substrate
(S).
•Therefore when E, S, and I are present, E can bind with
S to yield ES, or E can bind with I to yield EI. However E
cannot bind I and S simultaneously to yield a ternary EIS
complex i.e.

33. Figure 4
Competitive inhibition. (a) Kinetic scheme illustrating the binding of I to E. Note
that this is an expansion of Equation 5.11 that includes formation of the EI
complex. (b) Double-reciprocal plot. In competitive inhibition, Vmax remains
unchanged and Km increases. The black line labeled “Control” is the result in
the absence of inhibitor. The red lines are the results in the presence of
inhibitor, with the arrow showing the direction of increasing [I].

34. Non-competitive inhibition
•This form of inhibition occurs when an inhibitor molecule or ion can bind to a
second site on an enzyme surface (not the active site) but an inhibitor site.
•Noncompetitive inhibitors do not resemble the substrate.
•Inhibitor binding results in distortion or modification of the enzyme’s
conformation, which prevents product formation.
•The EIS ternary complex may either be totally or partially inactive in terms of
catalyzing the substrate to product reaction.
•In pure noncompetitive inhibition, with total inactivation, inhibitor binding does
not affect substrate binding.

35. Figure 5
Classic noncompetitive inhibition. (a) Kinetic scheme illustrating the binding of
I to E or ES. (b) Double-reciprocal plot. Vmax decreases, but Km remains the
same.

36. Uncompetitive Inhibition
•The inhibitor binds exclusively to the ES complex.
•Substrate binding results in a modification of the enzyme’s
conformation, which promotes the binding of the inhibitor.

37. Figure 6
Uncompetitive inhibition. (a) Kinetic scheme illustrating the binding of I to ES.
(b) Double-reciprocal plot. In uncompetitive inhibition, both Vmax and Km
decrease (i.e., the absolute values of both 1/Vmax and 1/Km obtained from the y
and x intercepts, respectively, increase). The ratio Km/ Vmax, the slope of the
lines, remains unchanged.

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Enzyme Inhibition

39. 39
Reference
Author(s) Title Year ISBN Publisher
Moran LA, Horton RA,
Scrimgeour G, Perry M
& Rawn D
Principles of
Biochemistry, 5th
edition
2011
978-
0321795793
Pearson