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ENZYMES
Prof.Dr. A. Sha Yaln
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Enzymes are highly specialized
proteins, they have evolved tocatalyze reactions in biological
systems and organisms.
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History of enzymes
1800s........... Digestion of meat by secretions of the
stomach.1850s........... Fermentation of sugar into alcohol by
yeast.
1850s........... Yeast extracts ferment sugar to alcohol.1926............ Isolation and crystallization of urease.
1930s........... Crystallization of pepsin and trypsin
Today...........Nearly two thousand different enzymesidentified, hundreds have been crystallized.
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Properties
Extraordinary catalytic power
High degree of specificity for substratesNo by-product formation
Function under mild conditions of temperature and
pH
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All enzymes are proteins (except for some
catalytic RNA molecules)The primary, secondary, tertiary and
quaternary structures of enzymes are all
essential to their catalytic activity
MW of enzymes range from 12,000 to >
1,000,000
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Some enzymes require an additional
chemical component other than their aminoacid residues for activity. Such additionalgroups are called cofactors
Cofactor(s) may be
one or more inorganic ions
a complex organic or metallo-organic molecule
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Fe2+
Cu2+
Zn2+
Mg2+
Mn2+
K+
Ni2+
Cytochrome oxidase
PeroxidaseDNA polymerase
Hexokinase
Arginase
Pyruvate kinase
Urease
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TPP
FADNAD
CoAPP
Co-B12THF
Transfer of aldehydes
Transfer of hydrogen atomsTransfer of hydride ion
Transfer of acyl groupsTransfer of amino groups
Transfer of H / alkyl groupsTransfer of 1 C atoms
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Prosthetic group covalently bound organic
molecule or metal ion
Coenzyme (Cosubstrate) tightly but notcovalently bound organic molecule
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A complete, catalytically active enzyme together
with its coenzyme and/or metal ions is called aholoenzyme.
The protein part of an enzyme is called the
apoenzyme or apoprotein.
HoloenzymeApo
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Enzyme Names
End in ase
Identifies substratesucrase reacts with sucrose
lipase - reacts with lipid
Describes function of enzyme
oxidase catalyzes oxidation
hydrolase catalyzes hydrolysis
Common names are still used
Pepsin, trypsin
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1. Oxidoreductases Transfer of electrons
2. Transferases Group-transfer reactions
3. Hydrolases Hydrolysis reactions
4. Lyases Addition to double bonds5. Isomerases Group-transfer in molecules
6. Ligases Bond forming reactions (ATP)
Classification
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ATP + Glucose ADP + Glucose-P
Trivial name: Hexokinase
Systematic name: ATP : glucose phosphotransferase
Classification no.: EC 2.7.1.12: Class name (transferase)
7: Subclass (phosphotransferase)
1: Sub-subclass (hydroxyl group as acceptor)1: Glucose as phosphate group acceptor
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Under biologically relevant conditions,
uncatalyzed reactions tend to be slow.
Enzymes provide a specific
environment within which a givenreaction is energetically more favorable.
Enzymes and catalysis
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Active siteEnzymes are proteins that catalyze chemical reactions.
Folding of the protein into itstertiary structure brings side-
chains of various amino acidsthat may be far apart in theprimary sequence into close
juxtaposition, forming anactive site.
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Enzyme
Active site
SWater P
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Reactive groups at the active site catalyze reactions by:
donating or withdrawing electrons
stabilizing or generating free radical intermediates
forming temporary covalent bonds (a transition state
intermediate)
There is high degree of specificity for the reaction catalyzed,i.e. an amino acid bound to pyridoxal phosphate may undergo:
isomerization, decarboxylation, transamination or side-
chain eliminationbut an enzyme will normally catalyze one of these reactions.
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1. Amino acids at the active site will make non-covalent interactions
between their side-chains and substrate molecule(s):
acidic groups (Asp, Glu) basic groups (Lys, His, Arg)
hydrophilic interactions with OH groups (Ser, Thr, Tyr)
hydrophilic interactions with SH groups (Cys) hydrophilic interactions with amide groups (Asn, Gln)
aromatic interactions (Phe, Tyr, Trp)
hydrophobic interactions (Ala, Leu, Ile, Val, Met, Pro)Amin2. Metal ions, carbohydrates and lipids bound to the enzyme may also
interact with substrates.3. Binding may result in considerable conformational change.
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There is specificity in binding to the active site
Because of multiple interactions in binding to the active site,enzymes can readily distinguish between isomers
C
C
OH
OH
H
CH2OH
C
COO-
CH3
NH3+H
D-glyceraldehyde D-alanine
C
C
H
OH
HO
CH2OH
C
COO-
CH3
H+H3N
L-glyceraldehyde L-alanine
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Enzymes can also distinguish between isomers
cis
trans
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Enzymes act by lowering the activation
energy of the reaction
initialexcited
final
+ enzyme
non-enzymicener
gylevel
They increase the speed at which equilibrium is achieved,but they do not alter the position of the equilibrium.
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Freeenergy
of
system
Progress of reaction
Activation energy barrier
Activation energy
of the catalyzed
reaction
Activationenergy
of the
uncatalyzed
reaction
Initial
state Overall
free-
energy
change
Final state at equilibrium
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Effect of pH
Enzymes have maximum activity at theiroptimum pH
Tertiary structure of enzyme must be maintained
Most enzymes have a narrow range of activity;
they loose their activity at low or high pHR groups of amino acids at the active site
determine pH profile
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Relative
rate
pH
2 4 6 8 10 12
Pepsin Glucose 6-phosphatase
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Effect of temperature
Enzymes will have very little activity at low
temperaturesReaction rate increases with temperature
Enzymes are most active at their optimum
temperatures (usually 37C in humans)
At high temperatures activity will be lost due
to denaturation of protein
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Relative
rate
Temperatureo
C
20 30 40 50 60 70
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Effect of substrate: [S]
Increasing substrate concentration increases
the rate of reaction (at constant enzymeconcentration)
Maximum activity is reached when all theenzyme combines with substrate
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Substrate concentration, M
Initial
rate
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Michaelis-Menten Equation
E + S ES E + P
E: enzyme
S: substrate
P: productES: enzyme-substrate complex
k1
k-1
k2
k-2
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1. Rate of formation of ES
Rate of formation = k1([Et] - [ES]) [S]
2. Rate of breakdown of ES
Rate of breakdown = k-1[ES] + k2[ES]
E + S ES E + P
k1 k2
k-1 k-2
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3. The steady state
Rate of formation = Rate of breakdown
k1([Et] - [ES]) [S] = k-1[ES] + k2[ES]
E + S ES E + P
k1 k2
k-1 k-2
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4. Separation of the rate constants
k1[Et] [S] = (k1 [S] + k-1 + k2) [ES]
[Et] [S]
[ES] =[S] + (k2 + k-1) / k1
E + S ES E + P
k1 k2
k-1 k-2
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5. Definition of initial velocity
vo = k2 [ES]
k2 [Et] [S]
vo =[S] + (k2 + k-1) / k1
E + S ES E + P
k1 k2
k-1 k-2
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6. Definition of Vmax and Km
Vmax = k2 [Et]
Km = (k2 + k-1) / k1
E + S ES E + P
k1 k2
k-1 k-2
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Michaelis-Menten EquationVmax [S]
vo =KM + [S]
vo= initial rate; Vmax= maximum rate;
KM = Michaelis-Menten constant
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Substrate concentration, M
Initial
rate
Vmax
1/2 Vmax
o
o
oo
o
oo
o o o o
KM
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Determination of Km and Vmax
0
0,2
0,4
0,6
0,8
1
0 200 400 600 800
[substrate]
relativeactivity maximum rate of reaction
when the enzyme is saturatedUnits: mol product / time
[Substrate] required to achieve VmaxUnits: mol substrate / L
Vmax
Km
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Lineweaver-Burk plot
1 / [substrate]
1/rate
1 / Vmax
-1 / Km
Slope= Km/ V max
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A high or low value of Km is relative to the physiological
range of concentration of substrate in cells
0
20
40
60
80
100
120
0 100 200 300 400
[substrate], mmol /L
rateofre
action,mol
/min
Km high compared with physiological range of [substrate]
large increase in rate of reactionfor a small increase in [substrate]
Km low compared with physiological range of [substrate]small increase in rate of reaction
for a large increase in [substrate]
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0
20
40
60
80
100
120
0 100 200 300 400
[substrate], mmol /L
rateofreaction,mo
l/min enzyme A
low Km
enzyme Bhigh Km
S
P
X
enzyme A
enzyme B
Two enzymes competing for substrate S
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Bisubstrate reactions
Single-displacement reactions
Double-displacement or ping-pongreactions
E
S1 S2
P
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Single displacement: (A + B C + D)
E + C + D
A
E
B
A
E
BA
E
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E
AX
E
AX
E
E E
B
E
A
BX
BX
Double displacement: (AX + B A + BX)
X
X
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Enzyme Inhibitors
cause loss of catalytic activity
change the protein structure of enzyme
may be competitive or noncompetitivesome are irreversible
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Enzyme inhibitors
Irreversible
Reversible Competitive
Non-competitive
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Enzyme inhibitors
irreversible bind to enzyme covalently
may undergo part of reaction transition state intermediate does not breakdown
reversible non-covalent (equilibrium) binding to enzyme
many are substrate analogues may be relatively unspecific
some inhibitors are used as drugs- mechanism-dependent (suicide) inhibitors
- highly specific for target enzyme- rational drug design
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Irreversible inhibition
E EII
Inactive
enzyme
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Competitive inhibition
E
SES
EII
E-Scomplex
Inactiveenzyme
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Competitive Inhibitors
have a structure similar to substrate
occupy the active site
compete with substrate for the active site
their effect is reversed by increasing
substrate concentration
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[substrate]
rateofreacti
on
1 / [substrate]
1/
rate
ofreacti
on
Vmax unchanged
Km increased
Competitive inhibition
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Noncompetitive Inhibitors
do not have a structure like substrate
bind to the enzyme but not to the active site
change the shape of enzyme and active site so thatsubstrate can not fit altered active site
their effect is not reversed by adding substrate
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Noncompetitive inhibition
ES
ES
E
E-S
complex
Inactiveenzyme
IS
S
I
I
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[substrate]
rateofreactio
n
1 / [substrate]
1/rateofreac
tion
Km unchanged
Vmax
decrease
d
Noncompetitive inhibition
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Uncompetitive inhibition
E
S
ES
E
E-S
complex
Inactive
enzyme S
I
I
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How can we determine whether aninhibitor is reversible or irreversible ?
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dialysis semi-permeable membrane
small moleculesequilibrateacross the membrane
proteins are too largeto cross the membrane
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semi-permeable membranedialysis
small moleculesequilibrateacross the membrane
proteins are too largeto cross the membrane
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semi-permeable membranedialysis
small moleculesequilibrateacross the membrane
proteins are too largeto cross the membrane
inhibitor removedactivity restored
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dialysis semi-permeable membrane
small moleculesequilibrateacross the membrane
proteins are too largeto cross the membraneinhibitor boundcovalently to protein
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semi-permeable membranedialysis
inhibitor not removedactivity not restored
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Enzymes are measured by their
catalytic activity; not by their mass.
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Factors affecting enzyme activity
pH of incubation or environment
temperature time of incubation
concentration of enzyme
concentration of substrate
covalent modification of enzyme
inhibitors and activators
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Information required for quantitative
enzyme measurement
1. Equation of the reaction
2. Analytical procedure3. Cofactor requirement
4. Substrate concentration dependency
5. Optimum pH6. Temperature dependency
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0
20
40
60
80
100
120
0 100 200 300 400 500 600 700 800
[substrate]
rateo
f
reactio
n
Excess substrate is used so that enzyme is saturated;limiting factor in product formation is [enzyme].
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Determining initial rates at increasing [enzyme]
Time
Progress
of
reaction
Initial
rates
E
3xE
2xE
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Enzyme activity vs. initial rate
Enzyme activity, units
Initial
rate
1
4 6 8 10 122
2
3
4
14
o
o
oo
oo
o
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Enzyme Activity (IU)
One unit of enzyme activity is defined as that
amount causing transformation of 1 mol ofsubstrate per minute under optimal conditions of
measurement.
The specific activity is the number of enzyme units
per milligram of protein.
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H2O
C CN
H
R1
O
N C
H H
R2
C
O
H
COOHH2N
C CN
H
R1
OH
H2N OH N C
H H
R2
C
O
COOH
H
In vitro:10 12 hours in 12 mol /L HCl at 105Crandom hydrolysis of peptide bonds
In vivo:
1 2 hours at 37C, specific bonds hydrolysed
An example of enzyme catalysis: serine proteases
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Histidine-57CH
HN
C
CH2
O
N N
Aspartate-102CH
HN
C
CH2-OOC
O
Serine-195CH
HN
C
CH2HO
O
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Bonds hydrolysed:
trypsin
esters of basic amino acids
chymotrypsin
esters of aromatic amino acids
elastase
esters of small neutral amino acids
-
Gly
Gly
Asp
-
+
peptide in groove on enzyme surface
trypsin
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Bonds hydrolysed:
trypsin
esters of basic amino acids
chymotrypsin
esters of aromatic amino acids
elastase
esters of small neutral amino acids
Gly
Gly
Ser
peptide in groove on enzyme surface
chymotrypsin
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Bonds hydrolysed:
trypsin
esters of basic amino acids
chymotrypsin
esters of aromatic amino acids
elastase
esters of small neutral amino acids
Val
Thr
Gly
peptide in groove on enzyme surface
elastase
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Lock-and-key Model
Enzyme binds the substrate in the active site
Only certain substrates can fit the active site
R-groups of amino acids forming the active siteaid substrate binding
Enzyme-substrate complex forms subtratechanges to product product is released fromthe enzyme
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E P2
P1
S S
E E
++
E + S ES E + P
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E E
E
Unfavorable orientation
Unfavorable proximity
Unfavorable orientation
Favorable proximity
Favorable orientation
Favorable proximity
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S
A
+S
Relaxed enzyme
molecule
Induced fit of enzyme
to the bound substrate
I d d fi d l
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Induced-fit model
Enzyme structure is flexible, not rigid
Enzyme and active site adjust theirshape to bind substrate
Both the enzyme and substrate
undergo conformational changeShape change
increases range of substrate specificityimproves catalysis during reaction
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P2
P1
S S +
E + S ES E + P
E EE+ S
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In the induced fit model;
when substrate binds the shape of the
enzyme adapts to the substrate.
P d /
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Proton donors/acceptors
carboxyl group (-COOH)
amino group (-NH2)
sulfhydryl group (-SH)
imidazole group
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Enzymes act in organized sequences
Some enzymes participating in cellular
metabolism are regulatory enzymes.
E1
A B C D
E2 E3
R l ti f E A ti it
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Regulation of Enzyme Activity
Inhibition
Allosteric regulation Covalent modification
Isoenzymes Synthesis/degradation
All i d l i
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Allosteric modulation
Increased
enzyme
activityM+
Decreased
enzyme
activity
M-
M
M
E
E
E
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v
[S]
+
Normal
rate -
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[substrate]
rateofreaction
Allosteric regulation is instantaneous activation by precursors inhibition by end-products
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06.12.2008 ASY/Enzymes 90substrate concentration
rateo
freaction
activation due to decreased cooperativity
inhibition due to increased cooperativity
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AA
C
C
A
B
B
S
A
B
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Aspartate Transcarbamoylase
Covalent modification
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Some enzymes are modified by
phosphorylation, glycosylation and otherprocesses.
These alterations effect enzyme activity, and
are involved in the regulation of enzymes.
Covalent modification
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H3PO4
H2O
ADP, H2O
ATP
serinephosphoserine
CH COHN
CH2
O
P
O
HO OH
CH2OH
CH COHN
CH2OH
CH COHN
serine
Covalent modification of an enzyme is
fast: time course of seconds minutes commonly in response to:
fast acting hormones (peptides, etc)
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CH2CH2
OH OH
CH2CH2
OOP P
Phosphorylase a(inactive)
Phosphorylase b(active)
Multiple forms of enzymes
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Multiple forms of enzymes
Many enzymes occur in more than one
molecular form in the same species, inthe same tissue, or even in the samecell.
Such multiple forms of enzymes arecalled isoenzymes or isozymes. Typical
examples are: lactate dehydrogenase(LDH), creatine kinase (CK)
Isoenzymes
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Isoenzymes
Enzymes catalysing the same reaction,
but differing in structure:
may have different charges at a given pH may have different affinity for substrate
may preferentially catalyse reaction in one direction
may differ in temperature sensitivity
may differ in inhibitor sensitivity
may differ in coenzyme specificity
They may be found in different tissues and in different
organelles
Lactate dehydogenase
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Lactate dehydogenase
CH3
C
COOH
O
CH3
CHOH
COOH
NAD+
NADH
NAD+
NADH
pyruvate lactate
pyruvate reduction in skeletal muscle
lactate oxidation in heart muscle
type 1 type 2 type 3 type 4 type 5
Separation of LDH isoenzymes
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Separation of LDH isoenzymes
The different isoenzymes have different charges,and can be separated by electrophoresis.
Type
1
Type2
Type3
Type4
Type5
Physiological control of enzyme activity
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06.12.2008 ASY/Enzymes 100
Physiological control of enzyme activity
Change in the rate of synthesis of the enzyme
(change in gene expression) slow: time course of hours or days
commonly in response to:
slow acting hormones (steroids)
long-term adaptation
Use of enzymes in medicine
8/8/2019 ASY Enzymes 08
101/102
06.12.2008 ASY/Enzymes 101
Use of enzymes in medicine
measurement of metabolites in plasma and urine
measurement of enzymes in plasma
assessment of tissue damage
physiological control of enzyme activity
use of enzyme inhibitors as drugs
Animations
8/8/2019 ASY Enzymes 08
102/102
Animations
http://www.kscience.co.uk/animations/model.swf
http://www.northland.cc.mn.us/biology/biology1111/
animations/enzyme.swf
http://cble.chem.uu.nl/biolip/SERPROTE.SWF
http://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swf
http://www.kscience.co.uk/animations/model.swfhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://cble.chem.uu.nl/biolip/SERPROTE.SWFhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://cble.chem.uu.nl/biolip/SERPROTE.SWFhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://www.kscience.co.uk/animations/model.swf