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55
5-1Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
The Behavior The Behavior of Proteins: of Proteins: EnzymesEnzymes
55
5-2Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme CatalysisEnzyme Catalysis• EnzymeEnzyme: a biological catalyst
• 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) Gibbs free energy (G) the relationship between entropy (S) and enthalpy (H), where
G = H - TS
55
5-3Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme CatalysisEnzyme Catalysis• For a reaction taking place at constant tempera
ture and pressure, e.g., in the body
the change in free energy is
• The change in free energy is related to the equilibrium constant, Keq, for the reaction by
A B
G° = H° - TS°
G° = RT ln Keq
55
5-4Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Activation Energy ProfileActivation Energy Profile
Progress of reaction
Fre
e e
nerg
y
reactants
products
Transition state
Activationenergy
G°
G°
Free energy change
55
5-5Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Activation Energy ProfileActivation Energy Profile• an enzyme alters the rate (kinetics) of a reaction, but
not its free energy change (thermodynamics) or position of equilibrium
Progress of reaction
Fre
e e
nerg
y
reactants
products
G°
Uncatalyzed reaction
Enzyme-catalyzedreaction
55
5-6Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme CatalysisEnzyme Catalysis• Consider the reaction
H2O2 H2O + O2
No catalyst
Platinum surface
Catalase
75.2 18.0
48.9 11.7
23.0 5.5
Activation energykJ/mol kcal/mol
Relativerate
1
4 x 1010
1 x 1021
55
5-7Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme KineticsEnzyme Kinetics• For the reaction
• the rate of reaction is given by
• where k is a proportionality constant called the specific rate constantspecific rate constant
• Order of reactionOrder of reaction: the sum of the exponents in the : the sum of the exponents in the rate equationrate equation
A + B P
Rate = [A]t
[B]t
[P]t
_ _= =
Rate = k[A]f[B]g
55
5-8Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme CatalysisEnzyme Catalysis• In an enzyme-catalyzed reaction
• substrate, Ssubstrate, S: the molecule(s) undergoing reaction
• active siteactive 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
55
5-9Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme CatalysisEnzyme Catalysis• Two models have been developed to describe
formation of the enzyme-substrate complex• lock-and-key modellock-and-key model: substrate binds to that portion
of the enzyme with a complementary shape• induced fit modelinduced fit model: binding of the substrate induces a
change in the conformation of the enzyme that results in a complementary fit
Also, H2O molecule play a much important role.
55
5-10Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme CatalysisEnzyme Catalysis• 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-nit
rophenylacetate
O2N OCCH3
O
+ H2O
chymo-trypsin
O2N O- CH3CO-+
pH > 7
Op-Nitrophenylacetate
p-Nitrophenolate
55
5-11Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ChymotrypsinChymotrypsin
Concentration of p-nitrophenylacetate (S)
Reacti
on
velo
cit
y (
V)
maximum velocity
55
5-12Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ATCaseATCase• Aspartate transcarbamylase (ATCase) catalyze
s this reaction
H2N-C-O-P-O-
O O
O-
CO2-
CH2
CH-CO2-
H3N++
H2N-C-NH-CH-CO2-
O
CO2-
CH2
+H3PO42-
ATCase
Carbamoylphosphate
Aspartate
N-Carbamoylaspartate
55
5-13Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ATCaseATCase
Concentration of aspartate (S)
React
ion
velo
city
(V
) maximum velocity
Note sigmoidal shape,which, as we will see, is one characteristic of allosteric enzymes
55
5-14Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme KineticsEnzyme Kinetics• Initial rate of an enzyme-catalyzed reaction
versus substrate concentration
Please see Fig. 5.6 (p156)
55
5-15Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Michaelis-Menten ModelMichaelis-Menten Model• for an enzyme-catalyzed reaction
• the rates of formation and breakdown of ES are given by these equations
• at the steady state
E + S ES Pk1
k-1
k2
k1[E][S] = k-1[ES] + k2[ES]
rate of formation of ES = k1[E][S]
rate of breakdown of ES = k-1[ES] + k2[ES]
55
5-16Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Michaelis-Menten ModelMichaelis-Menten Model• when the steady state is reached, the concentration
of free enzyme is the total less that bound in ES
• substituting for the concentration of free enzyme and collecting all rate constants in one term gives
• where KM is called the Michaelis constant
[E] = [E]T - [ES]
([E]T - [ES]) [S]
[ES] k-1 + k2
k1
= = KM
55
5-17Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Michaelis-Menten ModelMichaelis-Menten Model• it is now possible to solve for the concentration of
the enzyme-substrate complex in this way
• or alternatively
[ES] =[E]T [S]KM + [S]
[E]T [S] - [ES][S]
[ES]= KM
= KM[ES]
[E]T [S] = [ES](KM + [S])
[E]T [S] - [ES][S]
55
5-18Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Michaelis-Menten ModelMichaelis-Menten Model• in the initial stages, formation of product depends o
nly on the rate of breakdown of ES
• if substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T
• substituting k2[E]T = Vmax into the top equation gives
Vinit = k2[ES] = k2[E]T [S]KM + [S]
Vinit = Vmax = k2[E]T
Vmax [S]Vinit = KM + [S]
55
5-19Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Michaelis-Menten ModelMichaelis-Menten Model• when [S]= KM, the equation reduces to
Vmax [S]V =
KM + [S]=
Vmax [S]
[S] + [S]=
Vmax
2
(Presentation)
55
5-20Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Michaelis-Menten ModelMichaelis-Menten Model• it is difficult to determine Vmax experimentally
• the equation for a hyperbola
• can be transformed into the equation for a straight line by taking the reciprocal of each side
Vmax [S]V =
KM + [S](an equation for a hyperbole)
V1 =
KM + [S]
Vmax [S]=
KM [S]Vmax [S] Vmax [S]
+
V1 =
KM
Vmax [S] Vmax
+ 1
55
5-21Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Lineweaver-Burk PlotLineweaver-Burk Plot• which has the form y = mx + b, and is the formula for
a straight line
• a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax
• such a plot is known as a Lineweaver-Burk double reLineweaver-Burk double reciprocal plotciprocal plot
V1 =
Vmax
+ 1Vmax [S]
1
y m x + b
V1 =
KM •
55
5-22Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Lineweaver-Burk PlotLineweaver-Burk Plot
V1
[S]1
x intercept =
y intercept =1Vmax
-1KM
slope =KMVmax
55
5-23Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Significance of KSignificance of KMM and V and Vmaxmax
• 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 form product per mole of E per unit time
Acetylcholinesterase
Carbonic anhydrase
Catalase
Chymotrypsin
Turnover numbr
[(mol S)•(mol E)-1•s-1]
KM
(mol•liter-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
55
5-24Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Enzyme InhibitionEnzyme Inhibition• Reversible inhibitorReversible inhibitor: a substance that binds to
an enzyme to inhibit it, but can be removed• competitive inhibitorcompetitive inhibitor: binds to the active (catalytic)
site and blocks access to it by substrate• noncompetitive inhibitornoncompetitive inhibitor: binds to a site other than
the active site; inhibits by changing the conformation of the enzyme
• Irreversible inhibitorIrreversible inhibitor: inhibition cannot be reversed• usually involves formation or breaking of covalent
bonds to or on the enzyme
55
5-25Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Competitive InhibitionCompetitive Inhibition• substrate must compete with inhibitor for the active
site; more substrate is required to reach a given reaction velocity
• we can write a dissociation constant, KI for EI
E + S ES P+IEI
E+IEI KI =[E][I][EI]
55
5-26Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Competitive InhibitionCompetitive Inhibition
• 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
V1 =
KM
Vmax Vmax
+ 1
No inhibition
y b
S1•
m x +
y =
In the presence of a competitive inhibitor
V1 =
KM
Vmax Vmax+ 11 +[I]
KI S1
+ bm x
55
5-27Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Competitive InhibitionCompetitive Inhibition
V1
[S]1
x interceptsy intercept =1Vmax
slope =KMVmax
No inhibition
Competitiveinhibition
-1KM
-1
KM 1 +[I]
KI
KM
Vmax1 +
[I]
KIslope =
55
5-28Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Noncompetitive InhibitionNoncompetitive Inhibition• because the inhibitor does not interfere with binding
of substrate to the active site, KM is unchanged
• increasing substrate concentration cannot overcome noncompetitive inhibition
y = m x
In the presence of a noncompetitive inhibitor
V1 =
KM
Vmax Vmax+ 11 +
[I]
KI S1
+ b
1 +[I]
KI
V1 =
KM
Vmax Vmax
+ 1No inhibition
y b
S1•
m x +
55
5-29Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Noncompetitive InhibitionNoncompetitive Inhibition
V1
[S]1
x intercept y intercept =1Vmax
slope = KMVmax
No inhibition
Noncompetitiveinhibition
-1KM
KM
Vmax1 +[I]
KI
slope =
y intercept =1Vmax
1 +[I]
KI
55
5-30Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Allosteric EnzymesAllosteric Enzymes• AllostericAllosteric: Greek allo = other + steric = shape• Allosteric enzymeAllosteric enzyme: an oligomer whose biologic
al activity is affected by other substances binding to it• these substances change the enzyme activity by al
tering the conformation(s) of its 4° structure
• Allosteric effectorAllosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an• allosteric inhibitor• allosteric activator
• Aspartate transcarbamylase (ATCase)Aspartate transcarbamylase (ATCase)
55
5-31Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
H2N-C-OPO32-
O
CO2-
CH2
CH-CO2-H3N+
+
H2N-C-NH-CH-CO2-
O
CO2-
CH2
H3PO42-
ATCaseCarbamoylphosphate
Aspartate
N-Carbamoylaspartate
-O-P-O-P-O-P-O-CH2O
OHOH
HHHH
N
N
NH2
OO
O- O-
O
O-
O Series ofsteps
Cytidine triphosphate (CTP)
CTP inhibitsATCase!
55
5-32Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
-O-P-O-P-O-P-O-CH2O
OHOH
HHHH
N
N
NH2
OO
O- O-
O
O-
O
Cytidine triphosphate (CTP)
-O-P-O-P-O-P-O-CH2O
OHOH
HHHH
O
O- O-
O
O-
O
Adenosine triphosphate (ATP)
N
NN
N
NH2
an allosteric inhibitor of ATCase
an allosteric activator of ATCase
55
5-33Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ATCase - ATCase - an allosteric enzymean allosteric enzyme
[S]
React
ion
velo
city
(V
)
+ ATP (an allosteric activator)
+ CTP (an allosteric inhibitor
Control - no ATP or CTP
55
5-34Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
The Concerted ModelThe Concerted Model• Wyman, Monod, and Changeux - 1965• The enzyme has two conformations
• R (relaxed)R (relaxed): binds substrate tightly; the form active• T (tight)T (tight): binds substrate less tightly; the inactive for
m• 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
55
5-35Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Concerted ModelConcerted Model• the binding of substrate to one enzyme subunit facili
tates binding of a second substrate to a second enzyme subunit
• allosteric inhibitors bind to and stabilize the T (inactive) form
• allosteric activators bind to and stabilize the R (active) form
55
5-36Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Sequential ModelSequential Model• Koshland, Nemethy, and Filmer - 1966
• 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 induced-fit mechanism
55
5-37Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ZymogensZymogens• ZymogenZymogen: an inactive precursor of an enzyme;
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 residue
s cross linked by five disulfide (-S-S-) bonds• when it is secreted into the small intestine, the diges
tive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give -chymotrypsin
55
5-38Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ZymogensZymogens• the 15-unit polypeptide remains bound to -chymotr
ypsin by a single disulfide bond• -chymotrypsin catalyzes the hydrolysis of three of i
ts 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 f
rom chymotrypsinogen to -chymotrypsin result in changes in 2?and 3?structure as well.
• -chymotrypsin is enzymatically active because of its 2?and 3?structure, just as chymotrypsinogen was inactive because of its 2?and 3?structure
55
5-39Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
The Active SiteThe Active Site1. Which amino acid residues on the enzyme are i
n the active site and catalyze the reaction?
2. What is the spatial relationship of the essential amino acids residues in the active site?
3. What is the mechanism by which the essential amino acid residues catalyze the reaction?
• As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Lys or Arg
55
5-40Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ChymotrypsinChymotrypsin• Reaction with a model substrate
O2N OCCH3
O
O2N O-
CH3CO-O
p-Nitrophenylacetate
p-Nitrophenolate
Step 1 E +
E-OCH3
O+
Step 2 E-OCH3
O+ H2O E +
Enzyme
An acyl-enzymeintermediate
55
5-41Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ChymotrypsinChymotrypsin• DIPF inactivates chymotrypsin by reacting with
serine-195, which must be at the active site
Enz-CH2OH F-P-OCH(CH3)2
O
OCH(CH3)2
Diisopropylphospho-fluoridate
(DIPF)
+
Serine-195
Enz-CH2 O-P-OCH(CH3)2
O
OCH(CH3)2A labeled enzyme
(inactive)
55
5-42Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ChymotrypsinChymotrypsin• TPCK labels Histidine-57
Enz-CH2
N
NH
C6H5CH2-CH-C-CH2Cl
NH
O
Tsyl
N-Tosylamido-L-phenylethylchloromethyl ketone (TPCK)
(Tsyl = tosyl group)
+
Histidine-57
Enz-CH2
N
N
C6H5CH2-CH-C-CH2
NH
O
Tsyl
55
5-43Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
ChymotrypsinChymotrypsin• 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 definit
e arrangement of amino acids at the active site• in addition to His-57 and Ser-195, Asp-102 is also inv
olved in catalysis at the active site
• The mechanism by which chymotrypsin catalyzes the hydrolysis of amide bonds is shown in Figure 5.19
55
5-44Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Catalytic MechanismsCatalytic Mechanisms
Cys-SH Cys-S-
Lys-NH3+ Lys-NH2
Glu-CO2H Glu-CO2-
Ser-CH2OH Ser-CH2O-
His-CH2N
NH
H
His-CH2N
NH
Tyr OH Tyr O-
Proton Donor Proton AcceptorType of Group
sulfhydrylaminocarboxylhydroxyl
imidazole
phenol
+
55
5-45Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Catalytic MechanismsCatalytic Mechanisms• Lewis acid/base reactions
• Lewis acidLewis acid: an electron pair acceptor• Lewis baseLewis base: an electron pair donor
• Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes• carboxypeptidase A requires Zn2+ for activity
55
5-46Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Catalytic MechanismsCatalytic Mechanisms• Zn2+ of carboxypeptidase is complexed with
• 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 substitution
CC
O
NCH-CO2-H
R
OH
H
Zn(II)
Lewis acid
Lewis base
55
5-47Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Zn(II) of carboxypeptidase iZn(II) of carboxypeptidase is complexed withs complexed with
• 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 substitution
• (Please see p186)
55
5-48Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
CoenzymesCoenzymes• CoenzymeCoenzyme: a nonprotein molecule or ion that t
akes part in an enzymatic reaction and is regenerated for further reaction• metal ions• organic compounds, many of which are vitamins or
are metabolically related to vitamins
55
5-49Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Coenzyme Reaction TypeVitamin Precursor
Biotin
Coenzyme A
Flavin coenzymes
Lipoic acid
Nicotinamide coenzymes
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)
55
5-50Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Metal Ion Enzyme
Fe2+ or Fe3+
Cu2+
Zn2+
Mn2+
K+
Mg2+
Ni2+
Mo
Se
Peroxidase
Cytochrome oxidase
DNA polymerase
Hexokinase
Arginase
Pyruvate kinase
Urease
Nitrate reductase
Glutathione peroxidase
55
5-51Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
NADNAD++/NADH/NADH• Nicotinamide adenine dinucleotide (NAD+) is a
biological oxiding agent
a -N-glycoside bond
HH
H
O
HO OH
N
CNH2
O
+
The plus sign on NAD+represents the positive charge on this nitrogen Nicotinamide,
derivedfrom niacin;
-O-P-O-CH2
O
O
AMPH
55
5-52Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
NADNAD++/NADH/NADH• NAD+ is a two-electron oxidizing agent, and is
reduced to NADH
Ad
N
CNH2
O
++ H+ + 2e-
Ad
N
CNH2
OH H
NAD+
(oxidized form)NADH
(reduced form)
55
5-53Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
NADNAD++/NADH/NADH• NAD+ is involved in a variety of enzyme-
catalyzed oxidation/reduction reactions, two of which are
C
OH
H
C
O+ 2H+ 2e-
A secondary alcohol
A ketone
C H
O
+ H2O C OH
O
2H+ 2e-
An aldehyde A carboxylic acid
+
+ +
55
5-54Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
NADNAD++/NADH/NADH
N
CNH2
O
Ad
+
NAD+
N
CNH2
O
Ad
reduction
oxidation
H H
NADH
An electron pair is added to nitrogen
C
O
H
C
O
H
HE- B
HEB
2
3
4
1
55
5-55Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Pyridoxal PhosphatePyridoxal Phosphate
N
CH2OHHO
H3C
CHO
Pyridoxal
N
CH2OPO-HO
H3C
CH2NH2
Pyridoxamine phosphate
N
CH2OHHO
H3C
CH2NH2
Pyridoxamine
N
CH2OPO-HO
H3C
CHO
Pyridoxal phosphate
O
O-
O
O-
55
5-56Copyright (c) 1999 by Harcout Brace & CompanyAll rights reserved
Pyridoxal PhosphatePyridoxal Phosphate• Pyridoxal and pyridoxamine phosphates are inv
olved in the transfer of amino groups
-O2CCH2CH2CHCO2-
NH2
GlutamateCH3CCO2
-O
Pyruvate+
-O2CCH2CH2CCO2-
O
-Ketoglutarate
CH3CHCO2-
NH2
Alanine
+
transaminase,pyridoxal phosphate