ASY Enzymes 08

ENZYMES
Prof.Dr. A. Süha Yalçın
06.12.2008 ASY/Enzymes 1
Enzymes are highly specialized
proteins, they have evolved to
catalyze reactions in biological
systems and organisms.
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 enzymes
identified, hundreds have been crystallized.
Properties
¾ Extraordinary catalytic power
¾ High degree of specificity for substrates
¾ No by-product formation
¾ Function under mild conditions of temperature and
pH
¾ All enzymes are proteins (except for some
catalytic RNA molecules)
¾ The primary, secondary, tertiary and
quaternary structures of enzymes are all
essential to their catalytic activity
¾ MW of enzymes range from 12,000 to >
1,000,000
¾ Some enzymes require an additional
chemical component other than their amino
acid residues for activity. Such additional
groups are called cofactors
¾ Cofactor(s) may be
¾ one or more inorganic ions
¾ a complex organic or metallo-organic molecule
Fe2+ Cytochrome oxidase
Cu2+ Peroxidase
Zn2+ DNA polymerase
Mg2+ Hexokinase
Mn2+ Arginase
K+ Pyruvate kinase
Ni2+ Urease
TPP Transfer of aldehydes
FAD Transfer of hydrogen atoms
NAD Transfer of hydride ion
CoA Transfer of acyl groups
PP Transfer of amino groups
Co-B12 Transfer of H / alkyl groups
THF Transfer of 1 C atoms
• Prosthetic group – covalently bound organic
molecule or metal ion
• Coenzyme (Cosubstrate) – tightly but not
covalently bound organic molecule
A complete, catalytically active enzyme together
with its coenzyme and/or metal ions is called a
holoenzyme.
The protein part of an enzyme is called the
apoenzyme or apoprotein.
Apo Holoenzyme
Enzyme Names
z End in –ase
{ Identifies substrate
z sucrase – reacts with sucrose
z lipase - reacts with lipid
{ Describes function of enzyme
z oxidase – catalyzes oxidation
z hydrolase – catalyzes hydrolysis
z Common names are still used
{ Pepsin, trypsin
Classification
1. Oxidoreductases Æ Transfer of electrons

2. Transferases Æ Group-transfer reactions
3. Hydrolases Æ Hydrolysis reactions
4. Lyases Æ Addition to double bonds
5. Isomerases Æ Group-transfer in molecules
6. Ligases Æ Bond forming reactions (ATP)
ATP + Glucose ADP + Glucose-P
Trivial name: Hexokinase

Systematic name: ATP : glucose phosphotransferase
Classification no.: EC 2.7.1.1
2: Class name (transferase)
7: Subclass (phosphotransferase)
1: Sub-subclass (hydroxyl group as acceptor)
1: Glucose as phosphate group acceptor
Enzymes and catalysis
Under biologically relevant conditions,
uncatalyzed reactions tend to be slow.
Enzymes provide a specific
environment within which a given
reaction is energetically more favorable.
Active site
Enzymes are proteins that catalyze chemical reactions.
Folding of the protein into its

tertiary structure brings side-
chains of various amino acids
that may be far apart in the
primary sequence into close
juxtaposition, forming an
active site.
Water S P
Active site
Enzyme
Reactive groups at the active site catalyze reactions by:
¾ donating or withdrawing electrons
¾ stabilizing or generating free radical intermediates
¾ 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:
¾ isomerization, decarboxylation, transamination or side-
chain elimination
but an enzyme will normally catalyze one of these reactions.
1. Amino acids at the active site will make non-covalent interactions
between their side-chains and substrate molecule(s):
¾ acidic groups (Asp, Glu)
¾ basic groups (Lys, His, Arg)
¾ hydrophilic interactions with –OH groups (Ser, Thr, Tyr)
¾ hydrophilic interactions with –SH groups (Cys)
¾ hydrophilic interactions with amide groups (Asn, Gln)
¾ aromatic interactions (Phe, Tyr, Trp)
¾ hydrophobic interactions (Ala, Leu, Ile, Val, Met, Pro)Amin
2. Metal ions, carbohydrates and lipids bound to the enzyme may also
interact with substrates.
3. Binding may result in considerable conformational change.
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
H C O COO -
H C OH H C NH 3+
CH 2OH CH 3
D -glyceraldehyde D -alanine
H C O COO -
+H H
HO C H 3N C
CH 2OH CH 3
L -glyceraldehyde L -alanine
Enzymes can also distinguish between isomers
cis
trans
Enzymes act by lowering the activation
energy of the reaction
energy level
non-enzymic
+ enzyme
initial
excited
final
They increase the speed at which equilibrium is achieved,

but they do not alter the position of the equilibrium.
Activation energy barrier
Activation
energy
of the
uncatalyzed Activation energy
reaction of the catalyzed
reaction
Free
energy Initial
of state Overall
system free-
energy
change
Final state at equilibrium
Progress of reaction
Effect of pH
z Enzymes have maximum activity at their
optimum pH
z Tertiary structure of enzyme must be maintained
z Most enzymes have a narrow range of activity;
they loose their activity at low or high pH
z R groups of amino acids at the active site
determine pH profile
Pepsin Glucose 6-phosphatase
Relative
rate
2 4 6 8 10 12
pH
Effect of temperature
z Enzymes will have very little activity at low
temperatures
z Reaction rate increases with temperature
z Enzymes are most active at their optimum
temperatures (usually 37°C in humans)
z At high temperatures activity will be lost due
to denaturation of protein
Relative
rate
20 30 40 50 60 70
Temperature o C
Effect of substrate: [S]
¾ Increasing substrate concentration increases

the rate of reaction (at constant enzyme
concentration)
¾ Maximum activity is reached when all the
enzyme combines with substrate
Initial
rate
Substrate concentration, M
Michaelis-Menten Equation
E+S
k1 ES
k2 E+P
k-1 k-2
E: enzyme
S: substrate
P: product
ES: enzyme-substrate complex
k1 k2
E+S ES E+P
k-1 k-2
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]
k1 k2
E+S ES E+P
k-1 k-2
3. The steady state

Rate of formation = Rate of breakdown
k1([Et] - [ES]) [S] = k-1[ES] + k2[ES]
k1 k2
E+S ES E+P
k-1 k-2
4. Separation of the rate constants
k1[Et] [S] = (k1 [S] + k-1 + k2) [ES]
[Et] [S]
[ES] =
[S] + (k2 + k-1) / k1
k1 k2
E+S ES E+P
k-1 k-2
5. Definition of initial velocity
vo = k2 [ES]
k2 [Et] [S]
vo =
[S] + (k2 + k-1) / k1
k1 k2
E+S ES E+P
k-1 k-2
6. Definition of Vmax and Km
Vmax = k2 [Et]
Km = (k2 + k-1) / k1
Michaelis-Menten Equation
Vmax [S]
vo =
KM + [S]
vo= initial rate; Vmax= maximum rate;

KM = Michaelis-Menten constant
Vmax
o o o o o
o
o
Initial o
rate o 1/2 Vmax
o
o
KM Substrate concentration, M
Determination of Km and Vmax
1
Vmax
0,8
relative activity
maximum rate of reaction

0,6
when the enzyme is saturated
0,4 Units: mol product / time
0,2
0
0 200 400 600 800
[substrate]
Km
[Substrate] required to achieve ½ Vmax
Units: mol substrate / L
Lineweaver-Burk plot
1 / rate
Slope= Km / V max
1 / Vmax
1 / [substrate]
-1 / Km
A high or low value of Km is relative to the physiological
range of concentration of substrate in cells
rate of reaction, µmol /min
Km low compared with physiological range of [substrate]
120 small increase in rate of reaction
for a large increase in [substrate]
100
80
60
40
Km high compared with physiological range of [substrate]
large increase in rate of reaction
20
for a small increase in [substrate]
0
0 100 200 300 400
[substrate], mmol /L
enzyme A P
Two enzymes “competing” for substrate S S
rate of reaction, µmol /min enzyme B X
120 enzyme A
low Km
100
80
60
40 enzyme B
high Km
20
0
0 100 200 300 400
[substrate], mmol /L
Bisubstrate reactions
¾Single-displacement reactions
¾Double-displacement or ping-pong
reactions
S1 S2
P
Single displacement: (A + B C + D)
A B
A BA
E E E E +C+D
Double displacement: (AX + B A + BX)
AX A
AX X
E E E
B BX
X BX
E E E
Enzyme Inhibitors
¾cause loss of catalytic activity

¾change the protein structure of enzyme
¾may be competitive or noncompetitive
¾some are irreversible
Enzyme inhibitors
¾ Irreversible
¾ Reversible
• Competitive
• Non-competitive
Enzyme inhibitors
¾ irreversible
¾ bind to enzyme covalently
¾ may undergo part of reaction
¾ transition state intermediate does not breakdown
¾ reversible
¾ non-covalent (equilibrium) binding to enzyme
¾ many are substrate analogues
¾ may be relatively unspecific
¾ some inhibitors are used as drugs
- mechanism-dependent (suicide) inhibitors
- highly specific for target enzyme
- rational drug design
Irreversible inhibition
I
E I E Inactive
enzyme
Competitive inhibition
E-S
S E
S complex
I I
Inactive
E
enzyme
Competitive Inhibitors
¾ have a structure similar to substrate
¾ occupy the active site
¾ compete with substrate for the active site
¾ their effect is reversed by increasing
substrate concentration
Competitive inhibition
rate of reaction
1 / rate of reaction
Vmax unchanged
Km increased
[substrate] 1 / [substrate]
Noncompetitive Inhibitors
¾ do not have a structure like substrate

¾ bind to the enzyme but not to the active site
¾ change the shape of enzyme and active site so that
substrate can not fit altered active site
¾ their effect is not reversed by adding substrate
Noncompetitive inhibition
S E
E-S
S complex
E I
S
Inactive
I
S E enzyme
I
Noncompetitive inhibition
1 / rate of reaction
Vmax decreased
rate of reaction
[substrate] 1 / [substrate]
Km unchanged
Uncompetitive inhibition
S E-S
E S E
complex
Inactive
enzyme S E I
How can we determine whether an
inhibitor is reversible or irreversible ?
dialysis semi-permeable membrane
small molecules
equilibrate
across the membrane
proteins are too large

to cross the membrane
small molecules
equilibrate
across the membrane

small molecules
equilibrate
across the membrane

inhibitor removed
activity restored
small molecules
equilibrate
across the membrane

inhibitor bound
covalently to protein
inhibitor not removed

activity not restored
Enzymes are measured by their
catalytic activity; not by their mass.
Factors affecting enzyme activity
¾ pH of incubation or environment
¾ temperature
¾ time of incubation
¾ concentration of enzyme
¾ concentration of substrate
¾ covalent modification of enzyme
¾ inhibitors and activators
Information required for quantitative
enzyme measurement
1. Equation of the reaction

2. Analytical procedure
3. Cofactor requirement
4. Substrate concentration dependency
5. Optimum pH
6. Temperature dependency
Excess substrate is used so that enzyme is saturated;
limiting factor in product formation is [enzyme].
120
rate o f reaction
100
80
60
40
20
0
0 100 200 300 400 500 600 700 800
[substrate]
Determining initial rates at increasing [enzyme]
Initial
rates
3xE
Progress
of 2xE
reaction
Time
Enzyme activity vs. initial rate
4 o
o
3
o
o
Initial 2 o
rate
o
1 o
2 4 6 8 10 12 14
Enzyme activity, units
Enzyme Activity (IU)
One unit of enzyme activity is defined as that

amount causing transformation of 1 μmol of
substrate per minute under optimal conditions of
measurement.
The specific activity is the number of enzyme units
per milligram of protein.
An example of enzyme catalysis: serine proteases
H H O R2
H2N N C C N C C COOH
R1 H H O
H2O
H H O H R2
H2N N C C OH N C C COOH
R1 H H O
In vitro: 10 – 12 hours in 12 mol /L HCl at 105ºC

random hydrolysis of peptide bonds
In vivo: 1 – 2 hours at 37ºC, specific bonds hydrolysed
Bonds hydrolysed:
Trypsin:
esters of basic amino acids
Chymotrypsin: bond to be
esters of aromatic amino cleaved
lies over
acids
catalytic site
Elastase:
esters of small neutral
amino acids
substrate sits in a groove

on the enzyme surface
HN
- CH 2 CH
OOC Aspartate-102
C
O
HN
HO CH 2 CH Serine-195
C
O
HN
CH 2 CH Histidine-57
N N C
O
Bonds hydrolysed:
peptide in groove on enzyme surface
trypsin
chymotrypsin Gly
esters of aromatic amino acids +
Gly --
elastase
esters of small neutral amino acids
Asp
trypsin
Bonds hydrolysed:
trypsin
chymotrypsin Gly
esters of aromatic amino acids
Gly
elastase
Ser
chymotrypsin
Bonds hydrolysed:
trypsin
chymotrypsin Thr
esters of aromatic amino acids
elastase Val
Gly
elastase
Lock-and-key Model
¾ Enzyme binds the substrate in the active site

¾ Only certain substrates can fit the active site
¾ R-groups of amino acids forming the active site
aid substrate binding
¾ Enzyme-substrate complex forms Æ subtrate
changes to product Æ product is released from
the enzyme
P1
+ S S +
E E E P2
E + S ES E + P
Catalytic Efficiency of Enzymes
Proximity and orientation of the substrate in

relation to the catalytic group
Strain and distortion of the susceptible bond
(induced fit of the enzyme)
General acid-base catalysis
Covalent catalysis
Unfavorable orientation
Favorable proximity
E E
Unfavorable orientation Favorable orientation

Unfavorable proximity Favorable proximity
S
+
S
A
Relaxed enzyme Induced fit of enzyme
molecule to the bound substrate
Induced-fit model
¾Enzyme structure is flexible, not rigid
¾Enzyme and active site adjust their
shape to bind substrate
¾Both the enzyme and substrate
undergo conformational change
¾Shape change
{increases range of substrate specificity
{improves catalysis during reaction
P1
E + S E S E +
P2
E + S ES E + P
In the induced fit model;
when substrate binds the shape of the
enzyme adapts to the substrate.
Proton donors/acceptors
¾ carboxyl group (-COOH)

¾ amino group (-NH2)
¾ sulfhydryl group (-SH)
¾ imidazole group
Enzymes act in organized sequences
A B C D
E1 E2 E3
Some enzymes participating in cellular
metabolism are regulatory enzymes.
Regulation of Enzyme Activity
¾ Inhibition
¾ Allosteric regulation
¾ Covalent modification
¾ Isoenzymes
¾ Synthesis/degradation
Allosteric modulation
Increased
E M enzyme
M+ activity
M- E Decreased
enzyme
M activity
+
Normal
v rate -
[S]
rate of reaction
[substrate]
Allosteric regulation is instantaneous
¾ activation by precursors
¾ inhibition by end-products
rate of reaction activation due to decreased cooperativity
inhibition due to increased cooperativity
substrate concentration
A
B
SA A A
B
C
C
B
Aspartate Transcarbamoylase
Covalent modification
Some enzymes are modified by

phosphorylation, glycosylation and other
processes.
These alterations effect enzyme activity, and
are involved in the regulation of enzymes.
O
HO P OH
ADP, H2O O H3PO4
CH2OH ATP CH2 H2O

CH2OH
HN CH CO HN CH CO HN CH CO
serine phosphoserine serine
Covalent modification of an enzyme is

fast: time course of seconds – minutes
¾ commonly in response to:
¾ fast acting hormones (peptides, etc)
OH OH OP OP
CH2 CH2 CH2 CH2
Phosphorylase a Phosphorylase b
(inactive) (active)
Multiple forms of enzymes
¾Many enzymes occur in more than one

molecular form in the same species, in
the same tissue, or even in the same
cell.
¾Such multiple forms of enzymes are
called isoenzymes or isozymes. Typical
examples are: lactate dehydrogenase
(LDH), creatine kinase (CK)
Isoenzymes
Enzymes catalysing the same reaction,
but differing in structure:
¾ may have different charges at a given pH
¾ may have different affinity for substrate
¾ may preferentially catalyse reaction in one direction
¾ may differ in temperature sensitivity
¾ may differ in inhibitor sensitivity
¾ may differ in coenzyme specificity
They may be found in different tissues and in different

organelles
Lactate dehydogenase
pyruvate reduction in skeletal muscle

NAD+
NADH
CH3 CH3
C O CHOH
COOH COOH
+
pyruvate NAD lactate
NADH
lactate oxidation in heart muscle
type 1 type 2 type 3 type 4 type 5

Separation of LDH isoenzymes
Type 2
Type 3
Type 4
Type 5
+ Type 1 -
The different isoenzymes have different charges,

and can be separated by electrophoresis.
Physiological control of enzyme activity
Change in the rate of synthesis of the enzyme

(change in gene expression)
¾ slow: time course of hours or days
¾ commonly in response to:
¾ slow acting hormones (steroids)
¾ long-term adaptation
06.12.2008 ASY/Enzymes 100

Use of enzymes in medicine
¾ measurement of metabolites in plasma and urine

¾ measurement of enzymes in plasma
• assessment of tissue damage
¾ physiological control of enzyme activity
¾ use of enzyme inhibitors as drugs
06.12.2008 ASY/Enzymes 101

Animations
z http://www.kscience.co.uk/animations/model.swf
z http://www.northland.cc.mn.us/biology/biology1111/
animations/enzyme.swf
z http://cble.chem.uu.nl/biolip/SERPROTE.SWF
z http://www.stolaf.edu/people/giannini/flashanimat/
enzymes/allosteric.swf
06.12.2008 ASY/Enzymes 102

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ENZYMES

Prof.Dr. A. Süha Yalçın

1. Oxidoreductases Æ Transfer of electrons

Trivial name: Hexokinase

Folding of the protein into its

There is high degree of specificity for the reaction catalyzed,

Because of multiple interactions in binding to the active site,

They increase the speed at which equilibrium is achieved,

Final state at equilibrium

¾ Increasing substrate concentration increases

3. The steady state

4. Separation of the rate constants

k1[Et] [S] = (k1 [S] + k-1 + k2) [ES]

5. Definition of initial velocity

6. Definition of Vmax and Km

vo= initial rate; Vmax= maximum rate;

maximum rate of reaction

¾cause loss of catalytic activity

¾ do not have a structure like substrate

proteins are too large

proteins are too large

proteins are too large

proteins are too large

inhibitor not removed

1. Equation of the reaction

 One unit of enzyme activity is defined as that

In vitro: 10 – 12 hours in 12 mol /L HCl at 105ºC

substrate sits in a groove

¾ Enzyme binds the substrate in the active site

 Proximity and orientation of the substrate in

Unfavorable orientation Favorable orientation

¾ carboxyl group (-COOH)

inhibition due to increased cooperativity

 Some enzymes are modified by

CH2OH ATP CH2 H2O

serine phosphoserine serine

Covalent modification of an enzyme is

¾Many enzymes occur in more than one

They may be found in different tissues and in different

pyruvate reduction in skeletal muscle

lactate oxidation in heart muscle

type 1 type 2 type 3 type 4 type 5

The different isoenzymes have different charges,

Change in the rate of synthesis of the enzyme

06.12.2008 ASY/Enzymes 100

¾ measurement of metabolites in plasma and urine

06.12.2008 ASY/Enzymes 101

06.12.2008 ASY/Enzymes 102

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One unit of enzyme activity is defined as that

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Some enzymes are modified by