Download Chapter 5

Horton • Moran • Scrimgeour • Perry • Rawn
Principles of Biochemistry
Fourth Edition
Chapter 5
Properties of Enzymes
Prentice Hall c2002
Chapter 5 Copyright © 2006 Pearson Prentice
1
Hall, Inc.
Chapter 5 - Properties of Enzymes
• Catalyst - speeds up attainment of reaction
equilibrium
• Enzymatic reactions - 103 to 1017 faster than
the corresponding uncatalyzed reactions
• Substrates - highly specific reactants for
enzymes
Prentice Hall c2002
Chapter 5
2
Properties of enzymes (continued)
• Stereospecificity - many enzymes act upon
only one stereoisomer of a substrate
• Reaction specificity - enzyme product yields
are essentially 100% (there is no formation of
wasteful byproducts)
• Active site - where enzyme reactions take place
Prentice Hall c2002
Chapter 5
3
Prentice Hall c2002
Chapter 5
4
5.1 The Six Classes of Enzymes
1. Oxidoreductases (dehydrogenases)
• Catalyze oxidation-reduction reactions
Prentice Hall c2002
Chapter 5
5
2. Transferases
• Catalyze group transfer reactions
Prentice Hall c2002
Chapter 5
6
3. Hydrolases
• Catalyze hydrolysis reactions where water is
the acceptor of the transferred group
Prentice Hall c2002
Chapter 5
7
4. Lyases
• Catalyze lysis of a substrate, generating a
double bond in a nonhydrolytic, nonoxidative
elimination (Synthases catalyze the addition to
a double bond, the reverse reaction of a lyase)
Prentice Hall c2002
Chapter 5
8
5. Isomerases
• Catalyze isomerization reactions
Prentice Hall c2002
Chapter 5
9
6. Ligases (synthetases)
• Catalyze ligation, or joining of two substrates
• Require chemical energy (e.g. ATP)
Prentice Hall c2002
Chapter 5
10
5.2 Kinetic Experiments Reveal
Enzyme Properties
A. Chemical Kinetics
• Experiments examine the amount of product
(P) formed per unit of time (D[P] / Dt)
• Velocity (v) - the rate of a reaction
(varies with reactant concentration)
• Rate constant (k) - indicates the speed or
efficiency of a reaction
Prentice Hall c2002
Chapter 5
11
First order rate equation
• Rate for nonenzymatic conversion of substrate
(S) to product (P) in a first order reaction:
(k is expressed in reciprocal time units (s-1))
D[P] / Dt = v = k[S]
Prentice Hall c2002
Chapter 5
12
Second order reaction
• For reactions: S1 + S2
P1 + P2
• Rate is determined by the concentration
of both substrates
• Rate equation: v = k[S1]1[S2]1
Prentice Hall c2002
Chapter 5
13
Pseudo first order reaction
• If the concentration of one reactant is so high
that it remains essentially constant, reaction
becomes zero order with respect to that reactant
• Overall reaction is then pseudo first-order
v = k[S1]1[S2]0 = k’[S1]1
Prentice Hall c2002
Chapter 5
14
B. Enzyme Kinetics
• Enzyme-substrate complex (ES) - complex
formed when specific substrates fit into the enzyme
active site
E + S
ES
E+P
• When [S] >> [E], every enzyme binds a molecule
of substrate (enzyme is saturated with substrate)
• Under these conditions the rate depends only
upon [E], and the reaction is pseudo-first order
Prentice Hall c2002
Chapter 5
15
Fig 5.1 Effect of enzyme concentration [E]
on velocity (v)
• Fixed, saturating [S]
• Pseudo-first order
enzyme-catalyzed
reaction
Prentice Hall c2002
Chapter 5
16
Initial velocity (vo)
• Velocity at the beginning of an enzyme-catalyzed
reaction is vo (initial velocity)
• k1 and k-1 represent rapid noncovalent association
/dissociation of substrate from enzyme active site
• k2 = rate constant for formation of product from ES
E+S
Prentice Hall c2002
k1
k-1
ES
Chapter 5
k2
E+P
17
Fig 5.2 Progress curve for an
enzyme-catalyzed reaction
• The initial velocity (vo)
is the slope of the initial
linear portion of the
curve
• Rate of the reaction
doubles when twice as
much enzyme is used
Prentice Hall c2002
Chapter 5
18
5.3 The Michaelis-Menten Equation
• Maximum velocity (Vmax) is reached when an
enzyme is saturated with substrate (high [S])
• At high [S] the reaction rate is independent of
[S] (zero order with respect to S)
• At low [S] reaction is first order with respect to S
• The shape of a vo versus [S] curve is a
rectangular hyperbola, indicating saturation of
the enzyme active site as [S] increases
Prentice Hall c2002
Chapter 5
19
Fig 5.3 Plots of initial
velocity (vo) versus [S]
(a) Each vo vs [S] point is
from one kinetic run
(b) Michaelis constant (Km)
equals the concentration
of substrate needed for
1/2 maximum velocity
Prentice Hall c2002
Chapter 5
20
The Michaelis-Menten equation
• Equation describes vo versus [S] plots
• Km is the Michaelis constant
Vmax[S]
vo =
Km + [S]
Prentice Hall c2002
Chapter 5
21
A. Derivation of the
Michaelis-Menten Equation
• Derived from
(1) Steady-state conditions:
Rate of ES formation = Rate of ES decomposition
(2) Michaelis constant: Km = (k-1 + k2) / k1
(3) Velocity of an enzyme-catalyzed reaction
(depends upon rate of conversion of ES to E + P)
vo = k2[ES]
Prentice Hall c2002
Chapter 5
22
B. The Meanings of Km
• Km = [S] when vo = 1/2 Vmax
• Km @ k-1 / k1 = Ks (the enzyme-substrate
dissociation constant) when kcat << either k1 or k-1
• The lower the value of Km, the tighter the
substrate binding
• Km can be a measure of the affinity of E for S
Prentice Hall c2002
Chapter 5
23
Fig 5.4 Km and physiological
substrate concentrations
• Km values for enzymes
are typically just above
[S], so that the enzyme
rate is sensitive to small
changes in [S]
Prentice Hall c2002
Chapter 5
24
5.4 Kinetic Constants Indicate Enzyme
Activity and Specificity
• Catalytic constant (kcat) - first order rate
constant for conversion of ES complex to E + P
• kcat most easily measured when the enzyme is
saturated with S (region A on Figure 5)
• Ratio kcat /Km is a second order rate constant for
E+S
E + P at low [S] concentrations
(region B on Figure 5)
Prentice Hall c2002
Chapter 5
25
Fig 5.5 Meanings of kcat and kcat/Km
Prentice Hall c2002
Chapter 5
26
Table 5.1 Examples of catalytic constants
kcat(s-1)
Enzyme
Prentice Hall c2002
Chapter 5
27
Values of kcat/Km
• kcat/Km can approach rate of encounter of two
uncharged molecules in solution (108 to 109M-1s-1)
• kcat/Km is also a measure of enzyme specificity for
different substrates (specificity constant)
• rate acceleration = kcat/kn
(kn = rate constant in the absence of enzyme)
Prentice Hall c2002
Chapter 5
28
Prentice Hall c2002
Chapter 5
29
5.5 Measurement of Km and Vmax
Fig 5.6 The doublereciprocal LineweaverBurk plot is a linear
transformation of the
Michaelis-Menten plot
(1/vo versus 1/[S])
Prentice Hall c2002
Chapter 5
30
5.6 Kinetics of Multisubstrate Reactions
Fig 5.7 Notations for
bisubstrate reactions
(a) Sequential
(ordered or random)
Ordered
Random
Prentice Hall c2002
Chapter 5
31
Fig. 5.7 (b) Bisubstrate reactions
Prentice Hall c2002
Chapter 5
32
5.7 Reversible Enzyme Inhibition
• Inhibitor (I) binds to an enzyme and prevents
formation of ES complex or breakdown to E + P
• Inhibition constant (Ki) is a dissociation constant
EI
E+I
• There are three basic types of inhibition:
Competitive, Uncompetitive and Noncompetitive
• These can be distinguished experimentally by their
effects on the enzyme kinetic patterns
Prentice Hall c2002
Chapter 5
33
Fig 5.8 Reversible
enzyme inhibitors
(a) Competitive. S and I
bind to same site on E
(b) Nonclassical
competitive. Binding of
S at active site prevents
binding of I at separate
site. Binding of I at
separate site prevents S
binding at active site.
Prentice Hall c2002
Chapter 5
34
Fig 5.8 (cont)
(c) Uncompetitive.
I binds only to ES
(inactivates E)
(d) Noncompetitive.
I binds to either E or
ES to inactivate the
enzyme
Prentice Hall c2002
Chapter 5
35
A. Competitive Inhibition
• Inhibitor binds only to free enzyme (E) not (ES)
• Substrate cannot bind when I is bound at active site
(S and I “compete” for the enzyme active site)
• Vmax is the same with or without I (high S can still
saturate the enzyme even in the presence of I)
• Apparent Km (Kmapp) measured in the presence of I is
larger than KM (measured in absence of I)
• Competitive inhibitors usually resemble the substrate
Prentice Hall c2002
Chapter 5
36
Fig 5.9 Competitive inhibition.
(a) Kinetic scheme. (b) Lineweaver-Burk plot
Prentice Hall c2002
Chapter 5
37
Fig 5.10 Benzamidine competes with
arginine for binding to trypsin
Prentice Hall c2002
Chapter 5
38
B. Uncompetitive Inhibition
• Uncompetitive inhibitors bind to ES not to free E
• Vmax decreased by conversion of some E to ESI
• Km (Kmapp) is also decreased
• Lines on double-reciprocal plots are parallel
• This type of inhibition usually only occurs in
multisubstrate reactions
Prentice Hall c2002
Chapter 5
39
Fig 5.11 Uncompetitive inhibition
Prentice Hall c2002
Chapter 5
40
C. Noncompetitive Inhibition
• Noncompetitive inhibitors bind to both E and ES
• Inhibitors do not bind at the same site as S
• Vmax decreases
• Km does not change
• Inhibition cannot be overcome by addition of S
• Lines on double-reciprocal plot intersect on x axis
Prentice Hall c2002
Chapter 5
41
Fig 5.12 Noncompetitive inhibition
Prentice Hall c2002
Chapter 5
42
D. Uses of Enzyme Inhibition
Fig 5.13
Comparison of a substrate (a)
and a designed inhibitor (b)
for the enzyme purine
nucleoside phosphorylase
Prentice Hall c2002
Chapter 5
43
5.8 Irreversible Enzyme Inhibition
• Irreversible inhibitors form stable covalent bonds
with the enzyme (e.g. alkylation or acylation of an
active site side chain)
• There are many naturally-occurring and synthetic
irreversible inhibitors
• These inhibitors can be used to identify the amino
acid residues at enzyme active sites
• Incubation of I with enzyme results in loss of activity
Prentice Hall c2002
Chapter 5
44
Fig 5.14 Covalent complex with lysine residues
• Reduction of a Schiff base forms a stable
substituted enzyme
Prentice Hall c2002
Chapter 5
45
Inhibition of serine protease with DFP
• Diisopropyl fluorophosphate (DFP) is an organic
phosphate that inactivates serine proteases
• DFP reacts with the active site serine (Ser-195) of
chymotrypsin to form DFP-chymotrypsin
• Such organophosphorous inhibitors are used as
insecticides or for enzyme research
• These inhibitors are toxic because they inhibit
acetylcholinesterase (a serine protease that
hydrolyzes the neurotransmitter acetylcholine)
Prentice Hall c2002
Chapter 5
46
Fig 5.15
• Reaction of DFP with
Ser-195 of chymotrypsin
Prentice Hall c2002
Chapter 5
47
Affinity labels for studying
enzyme active sites
• Affinity labels are active-site directed reagents
• They are irreversible inhibitors
• Affinity labels resemble substrates, but contain
reactive groups to interact covalently with the
enzyme
Prentice Hall c2002
Chapter 5
48
Fig 5.16 Irreversible inhibition of
triose phosphate isomerase
• Bromo analog reacts with active site glutamate,
and reduction with NaBH4 stabilizes the adduct
Prentice Hall c2002
Chapter 5
49
5.9 Site-Directed Mutagenesis Modifies Enzymes
• Site-directed mutagenesis (SDM) can be
used to test the functions of individual
amino acid side chains
• One amino acid is replaced by another
using molecular biology techniques
• Bacterial cells can be used to synthesize
the modified protein
Prentice Hall c2002
Chapter 5
50
Practical applications of SDM
• SDM is also used to change the properties of
enzymes to make them more useful
• Subtilisin protease was made more resistant to
chemical oxidation by replacing Met-222 with
Ala-222 (the modified subtilisin is used in
detergents)
• A bacterial protease was made more heat
stable by replacing 8 of 319 amino acids
Prentice Hall c2002
Chapter 5
51
5.10 Regulation of Enzyme Activity
• Regulatory enzymes - activity can be reversibly
modulated by effectors
• Such enzymes are usually found at the first unique
step in a metabolic pathway (the first “committed”
step)
• Regulation at this step conserves material and
energy and prevents accumulation of intermediates
Prentice Hall c2002
Chapter 5
52
Two Methods of regulation
(1) Noncovalent allosteric regulation
(2) Covalent modification
• Allosteric enzymes have a second
regulatory site (allosteric site) distinct from
the active site
• Allosteric inhibitors or activators bind to this
site and regulate enzyme activity via
conformational changes
Prentice Hall c2002
Chapter 5
53
A. Phosphofructokinase-1 (PFK-1)
Is an Allosteric Enzyme
• PFK-1catalyzes an early step in glycolysis
(an ATP-generating pathway for glucose
degradation)
• Phosphoenol pyruvate (PEP), an intermediate
near the end of the pathway is an allosteric
inhibitor of PFK-1
• ADP is an allosteric activator of PFK-1
Prentice Hall c2002
Chapter 5
54
Regulation of glycolysis by PFK-1
• When the ratio [PEP]/[ADP] is high, flux through
glycolysis decreases at the PFK-1 step
• When the ratio of [PEP]/[ADP] is low, PFK-1 is
activated and glycolysis produces more ATP
from ADP
• Thus the concentrations of PEP and ADP act
allosterically through PFK-1 to regulate the
activity of the entire pathway
Prentice Hall c2002
Chapter 5
55
Fig 5.17 Reaction catalyzed by PFK-1
Prentice Hall c2002
Chapter 5
56
Fig 5.18 Phosphoenolpyruvate (PEP)
Prentice Hall c2002
Chapter 5
57
Effect of PEP and ADP on PFK-1
• Both PEP and ADP affect the binding of the substrate
fructose 6-phosphate (F6P) to PFK-1
• PFK-1 exhibits a sigmoidal (S-shaped) vo versus [S]
curve. This is due to the cooperativity of S binding.
• Increasing ADP concentrations lowers the apparent
Km for F6P without affecting Vmax (activates PFK)
• The allosteric inhibitor PEP raises the apparent Km
without changing Vmax
Prentice Hall c2002
Chapter 5
58
Fig 5.19 Plots of initial velocity versus
F6P for PFK-1
• ADP is an allosteric
activator of PFK-1
and lowers the
apparent Km without
affecting Vmax
• For a given F6P
concentration the vo
is larger in the
presence of ADP
Prentice Hall c2002
Chapter 5
59
Fig 5.20 Stereo views of PFK-1 from E. coli
• PFK-1 is a tetramer
of identical chains.
Products (yellow and
green), allosteric
activator ADP (red)
(a) Single chain
ribbon diagram
(b) Tetramer: 2 blue,
2 purple chains
Prentice Hall c2002
Chapter 5
60
B. General Properties of Allosteric Enzymes
1. Activities of allosteric regulator enzymes are
changed by inhibitors and activators (modulators)
2. Allosteric modulators bind noncovalently to the
enzymes that they regulate
3. Regulatory enzymes possess quaternary
structure
(continued next slide)
Prentice Hall c2002
Chapter 5
61
General properties of regulatory enzymes (cont)
4. Regulatory enzymes have at least one substrate
which has a sigmoidal vo versus [S] curve (positive
cooperativity of multiple substrate binding sites)
5. There is a rapid transition between the active (R)
and inactive (T) conformations
6. Substrates and activators may bind only to the R
state while inhibitors may bind only to the T state
Prentice Hall c2002
Chapter 5
62
Rapid transition exists between R and T states
• Addition of S increases concentration of the R state
• Addition of I increases concentration of the T state
• Activator molecules bind preferentially to R, leading
to an increase in the R/T ratio
Prentice Hall c2002
Chapter 5
63
Fig 5.21 Role of cooperativity of
binding in regulation
• Addition of modulators alters enzyme activity
• Activators can lower Km, inhibitors can raise Km
Prentice Hall c2002
Chapter 5
64
C. Two Theories of Allosteric Regulation
Concerted theory - (symmetry-driven theory).
• Only 2 conformations exist: R and T ( symmetry is
retained in the shift between R and T states)
• Subunits are either all R or all T
• R has high affinity for S, T has a low affinity for S
• Binding of S shifts the equilibrium toward all R state
• Binding of I shifts the equilibrium toward all T state
Prentice Hall c2002
Chapter 5
65
Sequential Theory (ligand-induced theory)
• A ligand may induce a change in the structure
of the subunit to which it binds
• Conformational change of one subunit may
affect the conformation of neighboring subunits
• A mixture of both R (high S affinity) and T (low
S affinity) subunits may exist (symmetry does
not have to be conserved)
Prentice Hall c2002
Chapter 5
66
Fig 5.22 Two models
(a) Concerted model:
subunits either all T state
or all R state
(b) Sequential model:
Mixture of T subunits and
R subunits is possible.
Binding of S converts
only that subunit from T
to R
Prentice Hall c2002
Chapter 5
67
Fig 5.23 Conformational changes
during O2 binding to hemoglobin
• Oxygen binding to Hb
has aspects of both the
sequential and
concerted models
Prentice Hall c2002
Chapter 5
68
D. Regulation by Covalent Modification
• Interconvertible enzymes are controlled by
covalent modification
• Converter enzymes catalyze covalent
modification
• Converter enzymes are usually controlled
themselves by allosteric modulators
Prentice Hall c2002
Chapter 5
69
Fig 5.24 General scheme for covalent
modification of interconvertible enzymes
Prentice Hall c2002
Chapter 5
70
Fig 5.25 Pyruvate dehydrogenase regulation
• Phosphorylation
stabilizes the inactive
state (red)
• Dephosphorlyation
stabilizes the active
state (green)
Prentice Hall c2002
Chapter 5
71
5.11 Multienzyme Complexes and
Multifunctional Enzymes
• Multienzyme complexes - different enzymes
that catalyze sequential reactions in the same
pathway are bound together
• Multifunctional enzymes - different activities
may be found on a single, multifunctional
polypeptide chain
Prentice Hall c2002
Chapter 5
72
Metabolite channeling
• Metabolite channeling - “channeling” of reactants
between active sites can occur when the product of
one reaction is transferred directly to the next active
site without entering the bulk solvent
• Can greatly increase rate of a sequence of reactions
• Can protect intermediates from solvent reactions
• Channeling is possible in multienzyme complexes
and multifunctional enzymes
Prentice Hall c2002
Chapter 5
73