Transcript Online Counseling Resource YCMOU ELearning Drive…

Online Counseling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
SEP-SBI091-CP1-04
Introduction
Programmes and Courses
SEP–SBI091- U01-CP1_04
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Credits
 Academic Inputs by
Sonali Alkari
Faculty YCMOU Nagpur Centre,
Faculty LAD college P.G. D of Biotechnology
Research officer Ankur Seeds Pvt Ltd
[email protected]
[email protected]
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How to Use This Resource

Counselor at each study center should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counseling.

Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.

Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.

Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.

Appear several times, for all the Self-Tests, available for this course.

Student can use handouts for last minutes preparation just before end
exam.
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Learning Objectives
 After studying this module, you should be
able to :
 Describe
Physical
and
Chemical
characteristics of enzymes
 Describe effect of pH,
 Describe
effect of Temperature and
others
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Factors affecting enzyme activity
1. Enzyme & Substrate concentration
2. Cofactors
3. Inhibitors and activators
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Enzyme concentration
 In order to study the effect of increasing the
enzyme concentration upon the reaction rate,
the substrate must be present in an excess
amount;
i.e.,
the
reaction
must
be
independent of the substrate concentration.
 Any change in the amount of product formed
over a specified period of time will be
dependent upon the level of enzyme present.
 The amount of enzyme present in a reaction is
measured by the activity it catalyzes. The
relationship
between
activity
and
concentration is affected by many factors such
as temperature, pH, etc.
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Enzyme concentration
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Substrate concentration-1
 With fixed enzyme concentration, an increase
of substrate will result at first in a very rapid
rise in velocity or rate of reaction.
 As substrate concentration continues to
increase, however the increase in the rate of
reaction begins to slow down until, with a large
substrate concentration , no further change in
velocity is observed.
 Enzyme-cataylzed reaction at varying substrate
concentration the active sites on the enzyme
molecules are not saturated by substrate and
thus the enzyme rate varies with substrate
concentration.
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Substrate concentration-2
 As the substrate molecule increases, the sites
are covered to a greater degree until at
saturation no more sites are available, the
enzyme is working at full capacity and now
the
rate
is
independent
of
substrate
concentration.
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Substrate concentration-3
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Substrate concentration-4
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Substrate concentration-5
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Substrate concentration-6
Why does the rate (velocity) of the
reaction increase then appear to reach
a plateau?
If the breakdown of the enzyme-substrate (ES) complex is rate
limiting the vt will plateau out.
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Substrate concentration-7
Hyperbolic curves are modelled by the
equation:
ax
y
b x
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Substrate concentration-8
The shape of the curve and the kinetics of
enzymes are modelled (described) by the
equation below:
V max [ S ]
v
Km  [ S ]
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Substrate concentration-9
1. What is Vmax?
Vmax is the maximum theoretical velocity of the
reaction.
2. What is Km?
Km is a measure of the affinity of an enzyme for a
given substrate. Functionally it is the [S] which
gives 0.5 x Vmax.
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Substrate concentration-10
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How do we calculate VMAX and KM?
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Problem!
 The curve never really reaches a
plateau so we cannot accurately
determine VMAX.
 If we cannot determine the VMAX then
we cannot determine the KM.
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How to measure these constants?
 Several methods are availble;





Lineweaver-Burk Plot
Eddie-Hofstee Plot
Hanes Plot
Computer programs
Other methods
(http://www.sbu.ac.uk/biology/enztech/dete
rmination.html)
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Lineweaver-Burk Plot
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Real Example
Source:http://wwwchem.csustan.edu/chem4400/kinetics.gif
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Cofactors
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Cofactors
Many enzymes need factors other than polypeptide
molecules for full activity.
Prosthetic groups
Organic molecules tightly bound to the enzyme.
Flavin adenine
dehydrogenase.
dinucleotide
(FAD)
of
succinate
Biotin of carboxylases e.g. pyruvate carboxylase.
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Cofactors
Coenzymes
Organic molecules loosely associated
the enzyme.
with
NAD+ or NADP+ in enzymes catalysing redox
reactions e.g. dehydrogenases.
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Cofactors
Metal ions
Zn2+ in carboxypeptidase A.
Cu2+ in cytochrome oxidase.
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Inhibitors, inducers and activators
Activators: increase the activity of an enzyme
e.g. ADP is an activator of PFK.
Inducers: switch on (induce) the synthesis of an
enzyme e.g. lactose induces the enzyme bgalactosidase.
Inhibitors: inhibit (decrease)
enzyme molecules.
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the
activity
of
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Inhibitors
There are three types of enzyme inhibitors:
1. Competitive inhibitors
2. Non-competitive inhibitors
3. Un-competitive inhibitors
Excess substrate can inhibit enzyme activity - in
this case substrate molecules bind to another
site causing inhibition.
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Competitive Inhibitors
As the name suggests competitive inhibitors
compete with the substrate at the substratebinding site.
The increase
unaffected.
the
Km
but
leave
the
Vmax
E.g. malonate is a competitive inhibitor of
succinate dehydrogenase.
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Examples
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Non-competitive Inhibitors
Non-competitive inhibitors bind at a site
away from the substrate-binding site. The
affect Vmax but do not affect Km.
E.g. the heavy metal ions Hg2+ and Pb2+
are non-competitive inhibitors.
 They are thought to bind to -SH groups
within the enzyme.
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Un-competitive Inhibitors
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Competitive and Non-competitive
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Enzyme Activity Depends on Ph-1
 Enzymes have an optimum pH (or pH range) at
which their activity is maximal; at higher or
lowerpH, activity decreases. This is not surprising.
 Amino acid side chains in the active site may act as
weak acids and bases with critical functions that
depend on their maintaininga certain state of
ionization, and elsewhere in the protein ionized side
chains may play an essential role in the interactions
that maintain protein structure.
 Removing a proton from a His residue, for example,
might eliminate an ionic interaction essential for
stabilizing the active conformation of the enzyme.
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Enzyme Activity Depends on pH-2
 A less common cause of pH sensitivity is
titration of a group on the substrate.
 The pH range over which an enzyme
undergoes changes in activity can provide a
clue to the type of amino acid residue
involved.
 A change in activity near pH 7.0, for example,
often reflects titration of a His residue.
 The effects of pH must be interpreted with
some caution, however.
 In the closely packed environment of a
protein, the pKa of amino acid side chains can
be significantly altered.
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Enzyme Activity Depends on pH-3
 For example, a near by positive charge can
lower the pKa of a Lysresidue, and a nearby
negative charge can increase it.
 Such effects sometimes result in a pKa that is
shifted by several pH units from its value in the
free amino acid.
 In the enzyme acetoacetate decarboxylase, for
example,one Lys residue has a pKa of 6.6
(compared with 10.5in free lysine) due to
electrostatic effects of nearby positive charges.
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Effect of Temperature
 Because of the protein nature of an enzyme
thermal denaturation of the enzyme protein
with increasing temperatures will decrease
the effective concentration of an enzyme and
consequently decrease the reaction rate.
 Up to perhaps 45 0C the predominant effect
will be an increase in reaction rate as
predicated by kinetic theory.
 Above 45 0C an opposing factor, namely
thermal denaturation will become increasingly
important, however, until at 55 0C rapid
denaturation
will
destroy
the
catalytic
function of the enzyme protein.
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What You Learn…
 The amount of enzyme present in a reaction is
measured by the activity it catalyzes.
 The enzyme activity is dependent of substrate and
enzyme concentration, cofactors, coenzymes and
inhibitors.
 Vmax is the maximum theoretical velocity of the
reaction.
 Km is a measure of the affinity of an enzyme for a
given substrate.
 The change in pH and temperature alter
the
structure of enzyme.
 There are different types of inhibitors; competitive,
non-competitive and uncompetitive.
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Critical Thinking Questions
1. What is the relationship between
enzyme and substrate concentration
and enzyme activity?
2. State different types of inhibitors?
3. What is the role of pH and
temperature in enzyme activity?
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Hints For Critical Thinking Question
1. Any change in the amount of product
formed over a specified period of time
will be dependent upon the level of
enzyme and substrate present,Vmax, Km.
2. competitive,
uncompetitive.
non-competitive
and
3. Denaturation of enzyme molecule take
place in extreme condition of pH and
temperature
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Study Tips:1
 Book1
 Title: Molecular Cell Biology
 Author: Harvey Lodish, David Baltimore
Publisher:Publishers: W. H. Freeman and
Company
 Book2
 Title: Principles of Biochemistry
 Author: AlbertL Lehninger
 Publisher:CBS Publishers & Distributors
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Study Tips:2
 Book3
 Title: Biochemistry
 Author: Lubert stryer
 Publishers: Freeman International
 Book4
 Title: Biochemistry
 Author: Keshav Trehan
 Publishers: Wiley Eastern
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Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia
http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia
(www.chem.qmul.ac.uk/iubmb/enzyme).
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End of the Presentation
Thank You!
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