Lecture6 enzymes in much detail

5
5-1
The Behavior
of Proteins:
Enzymes
Copyright (c) 1999 by Harcout Brace & Company
All rights reserved

5
• with the exception of some RNAs that catalyze their
own splicing (Chapter 8), all enzymes are proteins
• some enzymes are so specific that they catalyze the
reaction of only one stereoisomer, others catalyze a
family of similar reactions
• Gibbs free energy (G) the relationship between
5-2
Enzyme Catalysis
• Enzyme: a biological catalyst
entropy (S) and enthalpy (H), where
All rights reserved
G = H - TS

5
equilibrium constant, Keq, for the reaction by
5-3
Enzyme Catalysis
• For a reaction taking place at constant
temperature and pressure, e.g., in the body
the change in free energy is
• The change in free energy is related to the
All rights reserved
A B
G° = H° - TS°
G° = RT ln Keq

5
5-4
Activation Energy Profile
reactants
Transition state
Progress of reaction
Free energy
All rights reserved
Activation
energy
products
G°
G°
Free energy
change

5
Activation Energy Profile
• an enzyme alters the rate (kinetics) of a reaction, but
5-5
not its free energy change (thermodynamics) or
position of equilibrium
All rights reserved
Progres s of reaction
Free energy
re actants
products
G°
Uncatalyzed
re action
Enzyme -catalyzed
re action

5
5-6
Enzyme Catalysis
• Consider the reaction
H2O2 H2O + O2
No catalyst
Platinum surface
Catalase
All rights reserved
Activation energy
kJ/mol kcal/mol
75.2 18.0
48.9 11.7
23.0 5.5
Relative
rate
1
4 x 1010
1 x 1021

5
A + B P
Rate =
[A]
t
• Order of reaction: the sum of the exponents in the
5-7
Enzyme Kinetics
• For the reaction
• the rate of reaction is given by
• where k is a proportionality constant called the
specific rate constant
rate equation
All rights reserved
[B]
t
[P]
t
_ = _ =
Rate = k[A] f[B]g

5
• substrate, S: the molecule(s) undergoing reaction
• active site: the small portion of the enzyme surface
where the substrate(s) becomes bound by
noncovalent forces, e.g., hydrogen bonding,
electrostatic attractions, van der Waals attractions
5-8
Enzyme Catalysis
• In an enzyme-catalyzed reaction
All rights reserved

5
• Two models have been developed to describe
formation of the enzyme-substrate complex
• lock-and-key model: substrate binds to that portion
of the enzyme with a complementary shape
• induced fit model: binding of the substrate induces a
Also, H2O molecule play a much important role.
5-9
Enzyme Catalysis
change in the conformation of the enzyme that
results in a complementary fit
All rights reserved

5
• Chymotrypsin - catalyzes selective hydrolysis
of peptide bonds where the carboxyl is
contributed by Phe and Tyr
• it also catalyzes hydrolysis of the ester bond of p-nitrophenylacetate
5-10
Enzyme Catalysis
O2N OCCH3
p-Nitrophenylacetate O
All rights reserved
O
+ H2O
chymo-trypsin
pH > 7
O2N O- CH3CO- +
p-Nitrophenolate

5
5-11
Chymotrypsin
maximum velocity
Concentration of p-nitrophenylacetate (S )
Reaction velocity (V)
All rights reserved

5
5-12
ATCase
• Aspartate transcarbamylase (ATCase)
catalyzes this reaction
O-O
O
H2N-C-O-P-O-
All rights reserved
CO2
-
CH2
CH-CO2
-
+
H3 + N
H2N-C-NH-CH-CO2
-
O
CO2
-
CH2
2 -
H + 3PO4
ATCase
Carbamoyl
phosphate
Aspartate
N-Carbamoylaspartate

5
5-13
ATCase
maximum velocity
Concentration of aspartate (S )
All rights reserved
Note sigmoidal shape,
which, as we will see,
is one characteristic of
allosteric enzymes

5
5-14
Enzyme Kinetics
• Initial rate of an enzyme-catalyzed reaction
versus substrate concentration
All rights reserved
Please see Fig. 5.6 (p156)

5
5-15
Michaelis-Menten Model
• for an enzyme-catalyzed reaction
E + S ES P
• the rates of formation and breakdown of ES are
given by these equations
rate of formation of ES = k 1[E][S]
rate of breakdown of ES = k -1[ES] + k2[ES]
• at the steady state
All rights reserved
k1
k-1
k2
k1[E][S] = k-1[ES] + k2[ES]

5
• when the steady state is reached, the concentration
• substituting for the concentration of free enzyme
and collecting all rate constants in one term gives
5-16
of free enzyme is the total less that bound in ES
= = KM
• where KM is called the Michaelis constant
All rights reserved
[E] = [E]T - [ES]
([E]T - [ES]) [S ]
[ES]
k-1 + k2
k1

5
• it is now possible to solve for the concentration of
5-17
the enzyme-substrate complex in this way
[E]T [S ] - [ES][S ]
• or alternatively
All rights reserved
[ES] =
[E]T [S]
KM + [S]
[E]T [S ] - [ES][S ]
[ES]
= KM
= KM[ES]
[E]T [S ] = [ES](KM + [S ])

5
• in the initial stages, formation of product depends
Vinit = k2[ES] =
• substituting k2[E]T = Vmax into the top equation gives
5-18
only on the rate of breakdown of ES
• if substrate concentration is so large that the
enzyme is saturated with substrate [ES] = [E]T
All rights reserved
k2[E]T [S]
KM + [S]
Vinit = Vmax = k2[E]T
Vmax [S]
Vinit =
KM + [S]

5
5-19
• when [S]= KM, the equation reduces to
Vmax [S]
V =
KM + [S]
All rights reserved
=
Vmax [S]
[S] + [S]
=
Vmax
2
(Presentation)

5
Vmax [S]
V =
KM + [S]
• can be transformed into the equation for a straight
5-20
• it is difficult to determine Vmax experimentally
• the equation for a hyperbola
line by taking the reciprocal of each side
1 =
1 =
All rights reserved
(an equation for a hyperbole)
V
KM + [S ]
Vmax [S ]
=
KM [S]
+
Vmax [S ] Vmax [S ]
V
KM
+ 1
Vmax [S ] Vmax

5
Lineweaver-Burk Plot
• which has the form y = mx + b, and is the formula for
1 =
V
+ 1
1
KM •
• a plot of 1/V versus 1/[S] will give a straight line with
• such a plot is known as a Lineweaver-Burk double
5-21
a straight line
slope of KM/Vmax and y intercept of 1/Vmax
reciprocal plot
All rights reserved
Vmax
Vmax [S]
y m x + b

5
5-22
Lineweaver-Burk Plot
All rights reserved
1
V
1
[S]
x intercept =
y intercept =
1
Vmax
-1
KM
slope =
KM
Vmax

5
Significance of KM and Vmax
• KM is the dissociation constant for ES; the greater
the value of KM, the less tightly S is bound to E
• Vmax is the turnover number; moles of S that react to
5-23
form product per mole of E per unit time
Acetylcholines terase
Carbonic anhydrase
Catalase
Chymotrypsin
All rights reserved
Turnover numbr
[(mo l S)• (m ol E) -1•s -1]
KM
(m ol•lit er -1)
1.4 x 104
1.0 x 106
1.0 x 107
1.9 x 102
9.5 x 10-5
1.2 x 10-2
2.5 x 10-2
6.6 x 10-4

5
• Reversible inhibitor: a substance that binds to
an enzyme to inhibit it, but can be removed
• competitive inhibitor: binds to the active (catalytic)
site and blocks access to it by substrate
• noncompetitive inhibitor: binds to a site other than
reversed
• usually involves formation or breaking of covalent
5-24
Enzyme Inhibition
the active site; inhibits by changing the
conformation of the enzyme
• Irreversible inhibitor: inhibition cannot be
bonds to or on the enzyme
All rights reserved

5
Competitive Inhibition
• substrate must compete with inhibitor for the active
EI I + E + S ES P
5-25
site; more substrate is required to reach a given
reaction velocity
• we can write a dissociation constant, KI for EI
EI I + E KI =
All rights reserved
[E][I]
[EI]

5
1 =
V
KM
1
•
Vmax S
Vmax
In the presence of a competitive inhibitor
1 =
V
[I]
KI
1
• in a Lineweaver-Burk double reciprocal plot of 1/V
versus 1/[S], the slope (and the x intercept) changes
but the y intercept does not change
5-26
All rights reserved
+ 1
No inhibition
y m x +
b
y =
KM
1 + + 1
Vmax S
Vmax
m x + b

5
No inhibition
5-27
All rights reserved
1
V
1
[S]
x intercepts
slope =
y intercept =
1
Vmax
KM
Vmax
Competitive
inhibition
-1
KM
-1
KM 1 +
[I]
KI
KM
Vmax
1 +
[I]
KI
slope =

5
Noncompetitive Inhibition
• because the inhibitor does not interfere with binding
5-28
of substrate to the active site, KM is unchanged
• increasing substrate concentration cannot
overcome noncompetitive inhibition
1 =
1
S
•
In the presence of a noncompetitive inhibitor
1 =
V
KM
1
Vmax Vmax
y = m x
All rights reserved
+
1
1 +
[I]
KI
S
1 +
+ b
[I]
KI
V
KM
+ 1
Vmax Vmax
No inhibition
y m x +
b

5
Noncompetitive Inhibition
No inhibition
KM
Vmax
5-29
slope =
KM
Vmax
x intercept y intercept =
All rights reserved
1
V
1
[S]
1
Vmax
Noncompetitive
inhibition
-1
KM
1 +
[I]
KI
slope =
y intercept =
1
Vmax
1 +
[I]
KI

5
• Allosteric: Greek allo = other + steric = shape
• Allosteric enzyme: an oligomer whose
biological activity is affected by other
substances binding to it
• these substances change the enzyme activity by
• Allosteric effector: a substance that modifies
the behavior of an allosteric enzyme; may be
an
• allosteric inhibitor
• allosteric activator
5-30
Allosteric Enzymes
altering the conformation(s) of its 4° structure
• Aspartate transcarbamylase (ATCase)
All rights reserved

5
5-31
O
H2N-C-OPO3
2 -
CTP inhibits
ATCase !
O O
O
O
All rights reserved
CO2
-
CH2
CH-CO2
+
+ H3N -
H2N-C-NH-CH-CO2
-
O
CO2
-
CH2
H3PO4
2 -
ATCase
Carbamoyl
phosphate
Aspartate
N-Carbamoylaspartate
-O-P-O-P-O-P-O-CH2
O
N
NH2
N
H H
H H
OH OH
O- O-
O-
Series of
steps
Cytidine triphosphate (CTP)

5
an allosteric
activator of ATCase
NH2
an allosteric
inhibitor of ATCase
5-32
O O
O
O
-O-P-O-P-O-P-O-CH2
All rights reserved
O
N
NH2
N
H H
H H
OH OH
O- O-
O-
Cytidine triphosphate (CTP)
-O-P-O-P-O-P-O-CH2
O
N
H H
H H
OH OH
O
O-O
O-O
O- Adenosine triphosphate (ATP)
N
N N

5
ATCase - an allosteric enzyme
5-33
All rights reserved
[S]
+ ATP (an allosteric activator)
Control - no ATP or CTP
+ CTP (an allosteric inhibitor

5
• R (relaxed): binds substrate tightly; the form active
• T (tight): binds substrate less tightly; the inactive
form
• in the absence of substrate, most enzyme molecules
are in the T (inactive) form
• the presence of substrate shifts the equilibrium from
the T (inactive) form to the R (active) form
• in changing from T to R and vice versa, all subunits
change conformation simultaneously; all changes
are concerted
5-34
The Concerted Model
• Wyman, Monod, and Changeux - 1965
• The enzyme has two conformations
All rights reserved

5
facilitates binding of a second substrate to a second
enzyme subunit
• allosteric inhibitors bind to and stabilize the T
5-35
Concerted Model
• the binding of substrate to one enzyme subunit
(inactive) form
• allosteric activators bind to and stabilize the R
(active) form
All rights reserved

5
• the binding of substrate induces a conformational
change from the T form to the R form
• the change in conformation is induced by the fit of
the substrate to the enzyme, as per the induced-fit
model of substrate binding
• a change of one subunit from T to R makes the same
change easier in other subunits
• allosteric activation and inhibition also occur by the
5-36
Sequential Model
• Koshland, Nemethy, and Filmer - 1966
induced-fit mechanism
All rights reserved

5
• Zymogen: an inactive precursor of an enzyme;
residues cross linked by five disulfide (-S-S-) bonds
• when it is secreted into the small intestine, the
5-37
Zymogens
cleavage of one or more covalent bonds
transforms it into the active enzyme
• Chymotrypsinogen
• synthesized and stored in the pancreas
• a single polypeptide chain of 245 amino acid
digestive enzyme trypsin cleaves a 15 unit
polypeptide from the N-terminal end to give -
chymotrypsin
All rights reserved

5
chymotrypsin by a single disulfide bond
• -chymotrypsin catalyzes the hydrolysis of three of
its own peptide bonds to give -chymotrypsin
• -chymotrypsin consists of three polypeptide chains
joined by two of the five original disulfide bonds
• changes in 1?structure that accompany the change
from chymotrypsinogen to -chymotrypsin result in
changes in 2?and 3?structure as well.
• -chymotrypsin is enzymatically active because of
5-38
Zymogens
• the 15-unit polypeptide remains bound to -
its 2?and 3?structure, just as chymotrypsinogen
was inactive because of its 2?and 3?structure
All rights reserved

5
The Active Site
1. Which amino acid residues on the enzyme are
2. What is the spatial relationship of the essential
3. What is the mechanism by which the essential
enzyme of the digestive system that catalyzes
the selective hydrolysis of peptide bonds in
which the carboxyl group is contributed by Lys
or Arg
5-39
in the active site and catalyze the reaction?
amino acids residues in the active site?
amino acid residues catalyze the reaction?
• As a model, we consider chymotrypsin, an
All rights reserved

5
5-40
Chymotrypsin
• Reaction with a model substrate
O
All rights reserved
O
O2N OCCH3
O2N O-
O
CH3CO-
p-Nitrophenylacetate
p-Nitrophenolate
Step 1 E +
O
E-OCH3
+
Step 2 E-OCH3
+ H2O E +
Enzyme
An acyl-enzyme
intermediate

5
• DIPF inactivates chymotrypsin by reacting with
5-41
Chymotrypsin
serine-195, which must be at the active site
Enz -CH2OH F-P-OCH(CH3 ) 2
All rights reserved
O
OCH(CH3 ) 2
Diisopropylphospho-fluoridate
(DIPF)
+
Serine-195
O
Enz -CH2 O-P-OCH(CH3 ) 2
OCH(CH3 ) 2
A labe led enzyme
(inactive)

5
5-42
Chymotrypsin
• TPCK labels Histidine-57
All rights reserved
Enz -CH2
N
N
H
C6H5CH2 -CH-C-CH2Cl
NH
O
Tsyl
N-Tosylamido-L-phenylethyl
chloromethyl ketone (TPCK)
(Tsyl = tosyl group)
+
Histidine-57
Enz -CH2
N
N
C6H5CH2 -CH-C-CH2
NH
O
Tsyl

5
Chymotrypsin
• because Ser-195 and His-57 are required for activity,
they must be close to each other in the active site
• the results of x-ray crystallographic show the
definite arrangement of amino acids at the active
site
• in addition to His-57 and Ser-195, Asp-102 is also
5-43
involved in catalysis at the active site
• The mechanism by which chymotrypsin
catalyzes the hydrolysis of amide bonds is
shown in Figure 5.19
All rights reserved

5
Type of Group Donor Proton Acce ptor
Tyr OH Tyr O-Proton
5-44
Catalytic Mechanisms
Cys-SH Cys-S-
Lys-NH3
All rights reserved
+ Lys-NH2
Glu-CO2H Glu-CO2
-
Ser -CH2OH Ser -CH2O-
His-CH2
N
N
H
H
His-CH2
N
N
H
sulfhydryl
amino
carboxyl
hydroxyl
imidazole
phe nol
+

5
• Lewis acids such as Mn2+, Mg2+, and Zn2+ are
5-45
• Lewis acid/base reactions
• Lewis acid: an electron pair acceptor
• Lewis base: an electron pair donor
essential components of many enzymes
• carboxypeptidase A requires Zn2+ for activity
All rights reserved

5
• the imidazole side chains of His-69 and His-196 and
the carboxylate side chain of Glu-72
• it activates the carbonyl group for nucleophilic acyl
5-46
• Zn2+ of carboxypeptidase is complexed with
substitution
All rights reserved
C
C
O
N
CH-CO2 H -
R
O
H
H
Zn( II)
Lewis acid
Lewis base

5
Zn(II) of carboxypeptidase
is complexed with
• the imidazole side chains of His-69 and His-196
• it activates the carbonyl group for nucleophilic
5-47
and the carboxylate side chain of Glu-72
acyl substitution
• (Please see p186)
All rights reserved

5
• Coenzyme: a nonprotein molecule or ion that
takes part in an enzymatic reaction and is
regenerated for further reaction
• metal ions
• organic compounds, many of which are vitamins or
5-48
Coenzymes
are metabolically related to vitamins
All rights reserved

5
5-49
Coenzyme Reaction Type
All rights reserved
Vitamin
Pre cursor
Biotin
Coenzyme A
Flavin coenzymes
Lipoic acid
Nicotinamide
coe nzymes
Pyridoxal phosphate
Tetrahydrofolic acid
Thiamine
pyrophosphate
Carboxylation
Acyl transfer
Oxidation-reduction
Acyl transfer
Oxidation-reduction
Transamination
One-carbon
transfer
Aldehyde
transfer
-- -- -
Pantothenic acid
Riboflavin (B2)
-- -- -
Niacin
Pyridoxine (B6)
Folic acid
Thiamine (B1)

5
5-50
Metal Ion Enzyme
Fe2+ or Fe3+
Cu2+
Zn2+
Mg2+
Mn2+
K+
Ni 2+
Mo
Se
All rights reserved
Peroxidase
Cytochrome oxidas e
DNA polymerase
Hexokinas e
Arginase
Pyruvate k inase
Urease
Nitrate reductas e
Glutathione pe roxidase

5
• Nicotinamide adenine dinucleotide (NAD+) is a
5-51
NAD+/NADH
biological oxiding agent
The plus sign on NAD+
represents the positive
charge on this nitrogen Nicotinamide,
O
All rights reserved
O
a -N-glycoside
bond
H H
H
O
HO OH
N
CNH2
+
derived
from niacin;
-O-P-O-CH2
O
AMP
H

5
oxidation/reduction reactions, two of
5-53
NAD+/NADH
• NAD+ is involved in a variety of enzyme-catalyzed
which are
OH
C
H
O
All rights reserved
O
C
+ 2 H+ 2 e-
A secondary
alcohol
A ketone
C H
O
+ H2O C OH
2 H+ 2 e-
An aldehyde A carboxylic
acid
+
+ +

5
5-55
Pyridoxal Phosphate
CHO
HO CH2OH
N
H3C
Pyridoxal
HO CH2OH
All rights reserved
CHO
O
O-O
HO CH2OPO-H3C
CH2NH2
CH2OPO-H3C
HO N
Pyridoxamine phosphate
N
H3C
CH2NH2
Pyridoxamine
N
Pyridoxal phosphate
O-

5
• Pyridoxal and pyridoxamine phosphates are
5-56
Pyridoxal Phosphate
involved in the transfer of amino groups
-O2CCH2CH2CHCO2
All rights reserved
-
NH2
Glutamate
CH3CCO2
-
O
Pyruvate
+
-O2CCH2CH2CCO2
-
O
-Ketoglutarate
transaminase ,
pyridoxal phosphate
CH3CHCO2
-
NH2
Alanine
+

Lecture6 enzymes in much detail

Recomendados

Recomendados

Más contenido relacionado

Destacado

Destacado (20)

Similar a Lecture6 enzymes in much detail

Similar a Lecture6 enzymes in much detail (15)

Más de Vedpal Yadav

Más de Vedpal Yadav (11)

Lecture6 enzymes in much detail

Notas del editor