Chapter 8 Enzyme Catalysis
Homework II (cont’d): Chapter 8, Problems 4,
5, 7, 8, 9, 11 (the diagonal line with “Slope” is
the blue line), 12, 16, 17
Due October 25 (Wed).
1. Enzymes were among the first biological
macromolecules to be studied chemically
1.1 Much of the early history of biochemistry is
the history of enzyme research.
1.1.1 Biological catalysts were first
recognized in studying animal food digestion and
sugar fermentation with yeast (brewing and
wine making).
1.1.2 Ferments (i.e., enzymes, meaning in
“in yeast”) were thought (wrongly) to be
inseparable from living yeast cells for quite some
time (Louis Pasteur)
1.1.3 Yeast extracts were found to be able to
ferment sugar to alcohol (Eduard Buchner, 1897, who
won the Nobel Prize in Chemistry in 1907 for this
discovery).
1.1.4 Enzymes were found to be proteins (1920s to
1930s, James Sumner on urease and catalase, “all
enzymes are proteins”, John Northrop on pepsin and
trypsin, both shared the 1946 Nobel Prize in Chemistry).
1.1.5 Almost every chemical reaction in a cell is
catalyzed by an enzyme (thousands have been purified
and studied, many more are still to be discovered!)
1.1.6 Proteins do not have the absolute monopoly
on catalysis in cells. Catalytic RNA were found in the
1980s (Thomas Cech, Nobel Prize in Chemistry in 1989).
2. The most striking characteristics of enzymes are
their immense catalytic power and high specificity.
2.1 Enzymes accelerate reactions by factors of at least
a million.
2.1.1 Most reactions in biological systems do not
occur at perceptible rates in the absence of enzymes.
2.1.2 The rate enhancements (rate with enzyme
catalysis divided by rate without enzyme catalysis)
brought about by enzymes are often in the range of
107 to 1014)
2.1.3 For carbonic anhydrase, an enzyme
catalyzing the hydration of CO2 (H2O + CO2  HCO3+ H+), the rate enhancement is 107 (each enzyme
molecule can hydrate 105 molecules of CO2 per
second!)
2.2 Enzymes are highly specific both in the reaction
catalyzed and in their choice of substrates (i.e., reactants).
2.2.1 An enzyme usually catalyzes a single chemical
reaction or a set of closely related reactions (side reactions
leading to the wasteful formation of by-products rarely
occur).
2.2.2 Enzymes exhibit various degrees of specificity
in accord with their physiological functions (what of the
following?):
Low specificity: some peptidases, esterases,
and phosphatases.
Intermediate specificity: hexokinase, alcohol
dehydrogenases, trypsin.
Absolute or near absolute specificity: Many
enzymes belong to this group, and in extreme cases,
stereochemical specificity is exhibited (i.e., enantiomers
are distinguished as substrates or products).
2.3 Most enzymes are proteins.
2.3.1 Some enzymes require no other chemical
groups other than their amino acid residues for activity.
(e.g.)
2.3.2 Other enzymes require additional chemical
components called prosthetic groups (covalently bound)
(or cofactors).
2.3.3 Prosthetic groups could be inorganic metal
ions (e.g., Fe2+, Mg2+, Mn2+, Zn2+) or complex
organic or metalloorganic molecules called coenzymes.
2.3.4 A complete catalytically active enzyme
(including its prosthetic group) is called a holoenzyme.
2.3.5 The protein part of an enzyme (without its
prosthetic group) is called the apoenzyme.
2.3.6 Coenzymes often function as transient
carriers of specific (functional) groups during
catalysis.
2.3.7 Many vitamins, organic nutrients
required in small amounts in the diet, are
precursors of coenzymes.
3. Enzymes are classified by the reactions they catalyze
3.1 Trivial names are usually given to enzymes.
3.1.1 Many enzymes have been named by
adding the suffix “-ase” to the name of their
substrate or to a word or phrase describing their
activity (type of reaction).
3.2 Enzymes are categorized into six major classes
by international agreement.
3.2.1 The six major classes include
Oxidoreductases: catalyzing
oxidation-reduction reactions.
Transferases: catalyzing the transfer
of a molecular group from one molecule to
another.
3.2.1 (cont’d)
Hydrolases: catalyzing the cleavage
by the introduction of water.
Lyases: catalyzing reactions
involving removal of a group to form a double
bond or addition of groups to double bonds.
(e.g.?).
Isomerases: catalyzing reactions
involving intramolecular rearrangements.
Ligases: catalyzing reactions joining
together two molecules.
3.3 Each enzyme is given a systematic name
which identifies the reaction catalyzed (e.g.,
hexokinase is named as ATP:glucose
phosphotrasferase).
3.4 Each enzyme is assigned a four-digit number
with the first digit denoting the class it belongs,
the other three further clarifications on the
reaction catalyzed.
4. Enzymes, like all other catalysts, does not affect
reaction equilibria, only accelerate reactions.
4.1 Equilibrium constant (Keq’) of a reaction is related
to the free energy difference between the ground
states of the substrates and products (Go’)
 Go’ = -RTlnKeq’
Enzyme catalysis does not affect  Go’, thus not
Keq’.
4.2 The rate constant of a reaction (k) is related to the
free energy difference between the transition state and
the ground state of the substrate (G‡)
4.2.1 Transition state is a fleeting molecular
moment (not a chemical species with any significant
stability) that has the highest free energy during a
reaction.
4.2.2 An enzyme increases the rate constant of a
reaction (k) by lowering its G‡.
4.2.3 The combination of a substrate and an
enzyme creates a new reaction pathway whose transition
state energy is lower than that of the reaction in the
absence of energy.
5. Formation of an enzyme-substrate complex is the first
step in enzyme catalysis.
5.1 Substrates are bound to a specific region of an
enzyme called the active site.
5.1.1 Much of the catalytic power of enzymes
comes from their bringing substrates together in
favorable orientations in enzyme-substrate (ES)
complexes. (mutations that affect the on-rate, the offrate, kcat, …etc).
5.1.2 Most enzymes are highly selective in their
binding of substrates.
5.1.3 The active site usually takes up a relatively
small part of the total volume of an enzyme.
5.1.4 The active site is a three-dimensional entity
formed by groups that come from different parts of the
linear amino acid sequence.
5.1.5 Substrates are bound to enzymes by
multiple weak (noncovalent) attractions.
5.1.6 Active sites are clefts or crevices with a
generally nonpolar character (polar residues, when
present in the active site, usually participate in the
catalytic processes, thus called catalytic groups) (or
specificity of binding).
5.1.7 The active sites of some unbound
enzymes are complementary in shape to those of
their substrates (the lock-and-key metaphor, Emil
Fisher).
5.1.8 In many enzymes, the active sites have
shapes complementary to those of their substrates
only after the substrates are bound (the induced fit,
Daniel Koshland).
5.2 The existence of ES complexes has been shown
in a variety of ways.
5.2.1 The saturation effect: at a constant
concentration of an enzyme, the reaction rate
increases with increasing substrate concentrations
until a Vmax is reached.
5.2.2 ES complexes have been directly
observed by electron microscopy and X-ray
crystallography.
Dihydrofolate reductase, NADP+ (red),
tetrahydrofolate (yellow)
The lack of perfect complementarity is important to enzymatic
catalysis (induced fit) (not evident in this figure).
6. Binding energy is the major source of free energy
used by enzymes to lower the activation energies of
reactions.
6.1 Binding energy (Gb) is the energy derived from
enzyme-substrate interaction.
6.1.1 Formation of each weak interaction in the
ES complex is accompanied by a small release of free
energy.
6.1.2 Weak interactions are maximized when the
substrate is converted to the transition state.
6.1.3 The weak interactions that are formed
only in the transition state are those that make the
primary contribution to catalysis: Transition state
theory. In another words, the enzyme is evolved
(“designed”) to bind the transition state structure.
6.1.4 Catalytic antibodies can be formed by using
transition state analogs as immunogens (predicted by
William Jencks in 1969 and confirmed by Richard
Lerner and Peter Schultz in 1986). Problems: specific
chemical reaction, lack of substrate specificity. E.g.
Cutting DNA independent of or relative insensitive to
the flanking sequence.
6.2 The summation of the unfavorable (positive) G‡
and the favorable (negative) Gb results in a lower net
activation energy.
6.2.1 The requirement for multiple weak
interactions to drive catalysis is one reason why
enzymes (and some coenzymes) are so large. Therefore,
larger Gb.
6.3 Catalysis and specificity arise from the same
phenomenon.
6.3.1 Catalysis refers to the acceleration of the
reaction due to the involvement of enzymes.
6.3.2 Specificity refers to the ability of an enzyme
to discriminate between two competing substrates.
6.3.3 The same binding energy that provides
energy for catalysis also makes the enzyme specific.
(Gb-transition-state + Gb-substrate-general + Gb-substrate-specific)
But they are not always separable. It is intellectually
useful to make distinctions among these binding
energies, attributable to a particular group of atomic
interactions. They are often used in the combined
interpretation of kinetic, biochemical, genetic, and
structural data.
6.4 Binding energies can be used to overcome various
energy barriers that exist during catalysis.
6.4.1 Binding energy holds the substrates in the
proper orientation to react, thus overcome entropy
reduction (substrate recognition and complex
formation).
6.4.2 Formation of weak bonds between substrate
and enzyme also results in the unfavorable desolvation
of the substrate (requiring a binding energy of what
form? Nonspecific?).
6.4.3 Binding energy involving weak interactions
formed only in the reaction transition state helps to
compensate thermodynamically for any strain or
distortion that substrate must undergo to react (to
break or form a bond).
The interactions from the added groups contribute largely to
the stabilization of the transition state. Moreover, the rate can be
affected greatly by the interactions physically remote from the
covalent bond broken.
6.4.4 Weak interactions between substrates
and enzymes may generate conformational
changes on the enzyme, a phenomenon called
induced fit.
6.4.5 Induced fit may serve to bring specific
functional groups on the enzyme into the proper
orientation and position (alignment) to catalysis
(need to overcome the entropy increase).
6.4.6 Modern approaches combine multiple
theoretical (e.g., computational) and experimental
(e.g., mutagensis) approaches to the studies of
enzymes.
Reactions of an ester with a carboxylate group to form an anhydride
7. Properly positioned catalytic functional groups aid
bond cleavage and formation during enzyme catalysis.
7.1 The active sites of some enzymes contain amino
acid functional groups that can participate in the
catalytic process as proton donors or proton
acceptors--general acid-base catalysis.
7.1.1 Many biochemical reactions involve the
formation of unstable charged intermediates that
tend to break down rapidly to their constituent
reactant species.
7.1.2 Charged intermediates can often be
stabilized by transferring protons to or from the
substrate or intermediate to form a species that breaks
down to products more readily than to reactants.
Catalysis here means the facilitated (coordinated,
aligned) proton transfer.
7.1.3 General acid-base catalysis can provide a
rate enhancement on the order of 102 to 105.
7.2 Some enzymes accelerate reactions by forming
transient covalent intermediates with the substrates-covalent catalysis.
7.2.1 Amino acid side chains (e.g., Ser) and
prosthetic groups can function as nucleophiles in
forming covalent intermediates with substrates.
( leading to enzyme conformational changes)
7.2.2 The new pathway of reaction must have a
lower activation energy than the uncatalyzed one.
7.2.3 Free enzyme is always regenerated at the end
of the reaction.
7.3 Metal ions can participate in catalysis in several ways.
7.3.1 Metal ions can be tightly bound to the
enzyme or taken up from the solution along with the
substrate.
7.3.2 Metal ions (bound to the enzymes) can help
orient a substrate or stabilize charged reaction transition
states.
7.3.3 Metal ions can help activate substrates (how?
By polarizing the bond or creating a better nucleophile?)
7.3.4 Metal ions can mediate oxidationreduction reactions by reversibly change their
oxidation states (electron donor and acceptor).
7.3.5 Many enzymes have metal ions in their
active centers playing important roles in catalysis.
7.3.6 (Noncatalytic function) Metal ions can
also have structural purposes (e.g., Ca2+ binding
leads to conformational changes of calmodulin,
EF-hand, etc.). Regulation of enzymes is done
through regulation of their structures
(conformations).
7.4 An enzyme may use a combination of several
catalytic strategies to bring about a rate
enhancement.
7.4.1 Chymotrypsin uses both covalent
catalysis and general acid-base catalysis (details?)
8. Michaelis-Menton equation reflects the kinetic
behavior of many enzymes
8.1 Saturation effect was observed in enzyme catalysis
when plotting the initial velocity (Vo) against the
substrate concentration([S]).
8.1.1 Initial velocity (Vo) was measured at the
beginning of the enzyme-catalyzed reaction, when
substrate concentration can be considered constant ([S]
will decrease as the reaction progresses).
8.1.2 At relatively low concentrations of
substrate, Vo increases almost linearly with an increase
in [S].
If [S]<<Km,
8.1.3 At higher [S], Vo increases by smaller and
smaller amounts in response to the increase in [S].
8.1.4 Finally, a point (a plateau of maximum
velocity, Vmax) is reached beyond which there are only
vanishingly small increases in Vo with the increasing [S].
8.1.5 The ES complex was proposed to be a
necessary step in enzyme catalysis based on this kinetic
pattern.
8.2 The Michaelis-Menton equation was established to
account for the observed relationship between Vo and
[S].
8.2.1 A single-substrate, single-product reaction is
considered for simplicity.
8.2.2 At early times in the reaction, the
concentration of the product ([P]) is negligible and the
overall reaction can be written as (no reverse reaction)
k1
k2
E + S  ES  E + P
k-1
8.2.3 It is hypothesized (unnecessary unless in a
sequence of reactions) that the rate-limiting step (M-M
mechanism) in enzymatic reactions is the breakdown of
the ES complex to form the product and the free enzyme:
Vo = k2[ES]
8.2.4 The free enzyme concentration [E]=[Et]-[ES]
([Et] is the total enzyme concentration).
8.2.5 The amount of substrate bound by the
enzyme at any given time is negligible compared with the
total [S], because [S] is usually far greater than [Et]
([S]>>[Et], still true for low [S]); therefore, the free
substrate concentration is [S].
8.2.6 The rate of ES formation = k1([Et]-[ES])[S],
the rate of ES breakdown = k-1[ES]+k2[ES].
k1 is the substrate on-rate or the forward rate. k-1 is the
off-rate or the backward rate.
8.2.7 It is assumed that Vo reflects a steady state
in which [ES] is constant (steady-state kinetics),
k1([Et]-[ES])[S]= k-1[ES]+k2[ES]
k1[Et][S]
[ES]=----------------------k1[S]+k-1+k2
Because Vo = k2[ES],
k2k1[Et][S]
k2[Et][S]
Vmax[S]
Vo=------------------=-----------------------=--------------k1[S]+k-1+k2
[S]+(k-1+k2)/k1
[S]+Km
8.2.8 This is the Michaelis-Menton equation, and
Km =(k-1+k2)/k1, is the Michaelis-Menton constant.
8.2.9 The equation fits the observed curve very
well.
When [S] is very low (<<Km), Vo=Vmax[S]/Km
when [S] is very high (>>Km), Vo=Vmax
8.3 Km equals to the substrate concentration at which
the reaction rate is half its maximal value (Vo=Vmax/2).
8.3.1 This represents a practical (operational)
definition of Km.
8.4 Km and Vmax can be determined by varying the
substrate concentrations--The Michaelis-Menton
equation can be transformed by taking double reciprocal
of both sides into the following form:
1
Km
1
------ = ------------ + -------Vo
Vmax[S]
Vmax
8.4.1 A plot of 1/Vo versus 1/[S] yields a straight
line, which has an intercept of 1/Vmax on the 1/Vo axis, 1/Km on the 1/[S] axis, and a slope of Km/Vmax. The
double reciprocal plot is called the Lineweaver-Burk plot.
8.4.2 Alternatively, Km and Vmax can be obtained
by fitting the data into the Michaelis-Menton equation
using a computer program (software installed in modern
photospectrometers).
8.5 The real meaning of Km can change from one
enzyme to another.
8.5.1 A great many enzymes that may have quite
different reaction mechanisms (from what assumed
when establishing the equation, which is called
Micahelis-Menton mechanism, a single-substrate singleproduct two-step reaction, k2 is rate-limiting) follow
Michaelis-Menton kinetics: that is, exhibiting a
hyperbolic dependence of Vo on [S].
8.5.2 The actual meaning of Km depends on the
number and relative rates of the individual steps of the
reaction. (the non Michaelis-Menton mechanism).
8.5.3 In the two-step (Michaelis-Menton
mechanism) reaction, k2 is assumed rate-limiting, i.e.,
k2<<k-1, and Km reduces to k-1/k1, being the
dissociation constant (Ks) for the ES complex (the
inverse of the association or equilibrium constant, the
exchange rate of the substrate is so fast, ES is in
transitional equilibrium).
8.5.4 When Km=Ks, Km does reflects the affinity
between the substrate and enzyme in the ES complex.
8.5.5 k-1<<k2, Km=k2/k1. In this case, Km does
not reflect affinity between substrate and enzyme!
(Briggs-Haldane mechanism, k2/Km=(kcat/Km)=k1,
diffusion-controlled on-rate, ~108s-1M-1).
8.5.6 When k2 and k-1 are comparable,
Km=(k2+k-1)/k1, Km does not reflect the affinity
between substrate and enzyme!
8.5.7 When the reaction goes through
multiple steps after the formation of the ES
complex, Km then becomes a very complex
function of many rate constants! (Km does
not reflect substrate affinity in many cases)
(What’s the definitions of Kapp and kapp,
Km and kcat? Same! There are cases when
no real single ES exists, simply MichaelisMenton-like steady state kinetics).
app=apparent
8.6 The actual meaning of Vmax also varies from enzyme
to enzyme.
8.6.1 In the two-step (Michaelis-Menton) reaction,
k2 is assumed to be rate-limiting, Vmax=k2[Et]
8.6.2 The number of reaction steps and the identity
of the rate-limiting step(s) can vary from enzyme to
enzyme (thus Vmax varies).
8.7 A more general term kcat, instead of Vmax is usually
used.
8.7.1 kcat is defined as the limiting rate of any
enzyme-catalyzed reaction at saturating substrate
concentrations.
8.7.2 In a multiple reaction, kcat equals to (or less
than, upper bound) the rate constant of the clearly ratelimiting step if any (e.g., kcat=k2 in the ‘two-step’
Michaelis-Menton reaction)
8.7.3 When several steps are partially rate limiting,
kcat is a complex function of them all.
8.7.4 kcat is also called the turnover number,
equivalent to the number of substrate molecules
converted to product in a given unit of time on a single
enzyme molecule (at saturating substrate concentration).
8.8 The factor kcat/Km is generally the best kinetic
parameter to use in comparisons of catalytic efficiencies
of enzymes.
8.8.1 kcat is not satisfactory because two enzymes
having the same kcat may have very different rate
enhancements (catalyzed versus uncatalyzed, thus
different efficiencies). Also kcat reflects situations when
substrate concentration is saturating (most enzymes are
not normally saturated with substrates; under
physiological conditions the [S]/Km ratio is typically
between 0.01 and 1.0).
8.8.2 When [S]<<Km, Vo=(kcat/Km)[Et][S]
the ratio of the products of two reactions
=(kcat1/Km1)/(kcat2/Km2), others being equal, i.e., [Et]
and [S] are the same for the two reactions.
8.8.3 kcat/Km relates the reaction rate to the total
enzyme concentration and substrate concentration.
8.8.4 kcat/Km=k2/Km=k2k1/(k2+k-1)
8.8.5 Suppose the rate of formation of product (k2)
is much faster than the rate of dissociation of the ES
complex (k-1), then kcat/Km approaches k1 (Haldane).
8.8.6 The ultimate limit on the value of kcat/Km is
set by the rate of formation of the ES complex (k1, the
on-rate), which can not be faster than the diffusioncontrolled encounter of an enzyme and its substrate,
which is between 108 to 109 M-1s-1.
8.8.7 Many enzymes have a value of kcat/Km near
this range, attaining kinetic perfection.
8.8.8 Any further gain in catalytic rate can come
only by decreasing the time for diffusion (How could this
be realized? Directed kinetic binding through electrostic
field)
8.9 Many of the principles developed for the singlesubstrate systems may be extended to multisubstrate
systems.
8.9.1 The majority of enzymes involve two
substrates.
8.9.2 Most reactions obey Michaelis-Menton
kinetics when the concentration of one substrate is held
constant and the other is varied.
8.9.3 Reactions in which all the substrates bind to
the enzyme (to form a ternary complex) before the first
product is formed are called sequential mechanisms.
8.9.4 Sequential mechanisms are called ordered if
the substrates combine with the enzyme and the
products dissociate in an obligatory order.
8.9.5 A random mechanism implies no obligatory
order of combination or release.
8.9.6 Reactions in which one or more products are
released before all the substrates are added are called
ping-pong mechanisms.
8.9.7 Steady-state kinetics can be used to
distinguish the sequential and ping-pong mechanisms
(how?)
Sequential mechanism
Ping-pong mechanism
Sequential pathway
Ping-pong pathway
9. It is during the pre-steady state that the
individual rate constants may be observed.
9.1 A complete description of an enzyme-catalyzed
reaction requires direct measurement of the rates of
individual reaction steps.
9.1.1 Pre-steady state phase of a reaction is
generally very short: values of kcat usually lie
between 1 and 107s-1, thus measurement must be
made in a time range of 1 to 10-7s (rapid mixing and
sampling are needed).
9.1.2 Rapid mixing and sampling techniques
are available (e.g., the stopped-flow method).
Fast acylation
step
Slow deacylation
step
10. Enzymes are subject to reversible and
irreversible inhibition
10.1 The inhibition of enzymatic activity by
specific small molecules and ions is important.
10.1.1 It serves as a major control
mechanism in biological systems.
10.1.2 Many drugs and toxic agents act by
inhibiting enzymes.
10.1.3 Inhibition can be a source of insight
into the mechanism of enzyme action: residues
critical for catalysis can often be identified by
using specific (irreversible) inhibitors.
10.2 Reversible inhibition can usually be divided into
different types.
10.2.1 Reversible inhibitors bind to enzyme
noncovalently.
10.2.2 In competitive inhibition, the inhibitor
competes with the substrate for the active site (binding
of one prevents binding of the other, forming ES or EI
complexes but no ESI complexes, fig.).
10.2.3 Competitive inhibitors are often
compounds that resemble the substrates.
10.2.4 Vmax is not affected by the presence of a
competitive inhibitor (There is always some high
substrate concentration that will replace the inhibitor
from the enzyme’s active site).
10.2.5 Km is increased due to the presence of a
competitive inhibitor. Higher substrate concentration is
needed to achieve Vmax/2.
10.2.6 In noncompetitive inhibition, the inhibitor
binds to a site distinct from that (the active site) which
binds the substrate. (fig.)
10.2.7 Inhibitor binding does not affect substrate
binding and vice versa (i.e., inhibitor can bind to ES
complex, substrate can bind to EI complex).
10.2.8 The enzyme is inactivated when inhibitor is
bound (whether or not substrate is also present). (e.g.)
10.2.9 The apparent Vmax is lowered (due to the
concentration decrease of active enzymes)
10.2.10 Noncompetitive inhibition are only
observed with enzymes having two or more
substrates.
10.2.11 In uncompetitive inhibition, the
inhibitor binds only to the ES complex (unable to
bind to free enzyme). (fig.)
10.2.12 In an enzyme-catalyzed bisubstrate
reaction, an inhibition can be competitive,
noncompetitive, and uncompetitive at the same
time depending on the inhibitor used.
(Lineweaver-Burk plots).
Noncompetitive inhibition
a=1+[I]/KI
a’=1+[I]/K’I, K’I=[ES][I]/[ESI]
Uncompetitive inhibition
Mixed or noncompetitive inhibition, plots similar to
the sequential binding in ternary complexes
10.3 Irreversible inhibitors bind very tightly (covalently
or noncovalently) to the enzymes.
10.3.1 Many irreversible inhibitors modify critical
catalytic residues covalently, thus inactivating the
enzymes.
10.3.2 Diisopropylphosphofluoridate (DIPF, one
component of the toxic nerve gases) reacts with a critical
Ser residue on acetylcholineesterase.
10.3.3 Critical catalytic residues in the active site
can sometimes be identified using irreversible inhibitors
(DIPF on chymotrypsin).
10.3.4 A suicide inhibitor binds to the active site of
a specific enzyme, being converted to a very reactive
compound by the enzyme catalysis (mechanism-based),
then covalently modifies the enzyme, thus irreversibly
inhibits the enzyme activity. (e.g., after forming the
covalent intermediate, but lack of the ability to complete
the reaction.)
10.3.5 Suicide inhibitors could be very
effective drugs (rational drug design).
10.3.6 Transition state analogs act as potent
inhibitors for enzymes: transition-state analogs
usually bind to enzymes 102 to 106 times more
tightly than the normal substrates (strongly
supporting the transition-state theory for catalysis).
They are often a intermediate with a tetrahedral
structure.
F is a much better leaving group.
11. Enzyme activity is affected by pH
11.1 Each enzyme has an optimal pH or pH
range (where the enzyme has maximal activity).
11.1.1 Requirements for the catalytic
groups in the active site in appropriate
ionization state is a common reason for this
phenomenon.
11.1.2 Change of ionization state of surface
groups (which may affect the protein structure)
sometimes is responsible for this phenomenon.
11.1.3 In rare cases, it is the change of
ionization state of substrate that is responsible
for this phenomenon.
11.2 The pH range over which activity changes
can provide a clue to what amino acid residues
are involved.
11.2.1 This has to be treated with great
caution, because in a closely packed
environment of a protein, the pK values of
amino acid side chains can change significantly
(e.g., a nearby positive charge will increase the
pK value of a Lys residue, and a negative one
will decrease the pK!).
12. The molecular mechanisms of enzyme
catalysis is not easy to study.
12.1 A complete understanding of the catalytic
mechanism of an enzyme includes many aspects
including
12.1.1 Temporal sequence in which enzymebound reaction intermediates occur.
12.1.2 Structure of each intermediate and
transition state.
12.1.3 The rates of interconversion between
all intermediates.
12.1.4 The structural relationship of the
enzyme with each intermediate. (conformational
and chemical states of the enzyme).
11.1.5 The energetic contribution of all
reacting and interacting groups with respect to
the intermediate complexes and transition states
(utilization of binding energies).
11.1.6 There is probably no enzyme for
which the current understanding meets all these
requirements.
11.2 The catalytic mechanisms of chymotrypsin
is partially understood using a comprehensive
approach.
11.2.1 Pre-steady state kinetics studies
revealed that the hydrolysis of pnitrophenylacetate by chymotrypsin, as
measured by release of p-nitrophenol (a colored
product) consists of a fast phase (acylation-covalent catalysis) and a slow phase
(deacylation). In hydrolysis of proteins, the
deacylation step is rate-limiting!
11.2.2 Two catalytic residues (Ser195 and
His57) were identified by chemical
modifications (using DIPF and TPCK
respectively).
11.2.3 X-ray structure determination of
the enzyme revealed the presence of Ser195
(function as a nucleophile to attack the
carbonyl carbon) and His57 (function as
proton acceptor first and then as proton donor
in the catalytic process--general acid-base
catalysis) at the active site of the chymotrypsin.
11.2.4 Ser195 and His57 form a catalytic triad
with the buried Asp102 residue (which stabilizes
the positive charge formed on the His57 residue,
that in turn prevents the development of a very
unstable positive charge on Ser195 hydroxyl,
thus making Ser195 a more effective
nucleophile).
11.2.5 At the transition state, the carbonyl
oxygen acquires a negative charge, which is
stabilized by hydrogen bonds formed from
groups (which residues?) in an oxyanion hole at
the active site.
11.3 Reasons for the markedly different specificity
of chymotrypsin, trypsin, and elastase were
revealed by structure determination of the three
enzymes.
11.3.1 Chymotrypsin has a nonpolar pocket
serving as the binding site for the aromatic or
bulky nonpolar side chains.
11.3.2 Trypsin has an Asp residue replacing a
Ser residue in the binding site, and so, recognize
only residues with positively charged side chains
(Lys and Arg).
11.3.3 Elastase has two much bulkier Val and
Thr replacing two Gly residues at the entrance of
the pocket, and so, only smaller side chains can
bind.
木糖
Glycolysis: 糖原酵解
13. The enzymatic activity of some enzymes are
precisely and tightly regulated in living organisms
to meet physiological requirements.
13.1 Allosteric enzymes (similar to hemoglobin)
are regulated by reversible, noncovalent binding
of modulators (often being metabolites).
13.1.1 Feedback inhibition, i.e., building up
of a pathway’s end product ultimately slows the
entire pathway, is often realized through allosteric
enzymes.
13.1.2 The enzyme catalyzing the first step of
a synthetic pathway is often an allosteric enzyme.
13.1.3 For example, threonine dehydratase
in the Ile synthesis pathway, and aspartate
transcarbamoylase (ATCase, the best understood
allosteric enzyme) in the pyrimidine nucleotide
synthesis pathway.
13.1.4 The modulators usually bind not to
the active site but to another specific regulatory
site.
13.1.5 The enzyme activity varies when the
concentration of the modulators vary, hence the
synthesis pathway is open only when the end
product is lacking (closed when the end product is
abundant).
13.2 Allosteric enzymes do not follow the
Michaelis-Menton kinetics.
13.2.1 They do exhibit saturation effect, but
do not show a hyperbolic curve when plotting Vo
against [S].
13.2.2 The substrate concentration at which
Vo is half maximal is referred as K0.5 (which is
not Km!)
13.2.3 Homotropic enzymes show sigmoidal
saturation curve when plotting Vo against [S],
where the substrate itself is a positive
(stimulatory) modulator for a multiple-subunit
cooperative enzyme system.
13.2.4 Heterotropic enzymes have substratesaturation curves in various shapes, where the
modulator is a metabolite other than the
substrate.
13.2.5 A positive modulator may make the
curve more hyperbolic-like (still sigmoidal)
(with a decreased K0.5, but no change on Vmax).
13.2.6 A negative modulator may make the
curve more sigmoidal (with increased K0.5 and
unchanged Vmax).
13.2.7 In a less common type of
modulation, Vmax is changed with K0.5 almost
constant.
13.3 Two models have been proposed to explain
the cooperativity phenomenon of some
homotropic allosteric enzymes.
13.3.1 They are the same as the two
models proposed to explain the cooperative
oxygen binding process of hemoglobin: the
sequential model and the concerted model.
13.4 The activity of many enzymes are
regulated by reversible covalent modifications.
13.4.1 Phosphorylation, the most
common reversible covalent modification, is a
highly effective means of switching the activity
of target enzymes.
13.4.2 Protein kinases catalyze the
transfer of a phosphate group from an ATP
molecule to the side chains of Ser, Thr, or Tyr
residues in proteins.
13.4.3 Protein phosphatases catalyze the
hydrolysis of phosphoryl groups attached to
proteins, thus reversing the effects of kinases.
13.4.4 The degree of specificity of protein
kinases varies. Some catalyze the phosphorylation
of many different target proteins (at sites of
conserved sequences, e.g., protein kinase A
recognizes a conserved sequence made of Arg-ArgX-Ser-Z or Arg-Arg-X-Thr-Z). Some
phosphorylate a single protein or several closely
related ones.
13.4.5 Phosphorylation is a highly effective
means of controlling the activity of proteins for
structural, thermodynamic, and kinetic reasons.
(elaborate please).
Glucose (red), AMP (dark blue, allosteric
activator), pyridoxal phosphate (PLP, B6
derivative, light blue), and ser14 (yellow)
Glycogen phosphorylase
13.5 Stimulatory or inhibitory proteins, as well as
small molecules, regulate the activity of enzymes.
13.5.1 Most effects of cyclic AMP (formed
when many hormones interact with their receptors
on the cell surface, G-protein signal transduction
pathway) in eukaryotic cells are mediated by the
activation of a single protein kinase, called protein
kinase A (PKA).
13.5.2 Cyclic AMP activates PKA by binding
to its two regulatory subunits, thus relieving the
two catalytic subunits, which then become active.
13.5.3 A pseudosubstrate sequence (a
segment of peptide) on the regulatory subunits
occupies the active site of the two catalytic
subunits, thus inhibiting their activity.
13.5.4 Calmodulin activates many proteins
inside a cell when calcium levels rise.
13.5.5 Blood clotting is markedly accelerated
by antihemophilic factor, a protein that enhances
the activity of a serine protease.
13.6 Many enzymes are activated by specific
proteolytic cleavage.
13.6.1 These enzymes are synthesized as
inactive precursors called zymogens.
13.6.2 They are activated by cleavage of one
or several specific peptide bonds.
13.6.3 Proteolytic activation, in contrast
with allosteric control and reversible covalent
modification, can occur just once in the life of the
enzyme molecule.
13.6.4 The digestive enzymes that hydrolyze
proteins are synthesized as zymogens in the
stomach and pancreas.
13.6.5 Blood clotting is mediated by a
cascade of proteolytic activation.
13.6.6 Some protein hormones are
synthesized as inactive precursors (e.g., insulin is
derived from proinsulin by proteolytic removal
of a peptide).
13.7 Amount of some enzymes are increased
when certain inducers (often enzyme substrates)
are present in the cells.
13.7.1 This is often seen in bacterium cells.
13.7.2 The presence of lactose in a culture
medium induces a large increase in the amount
of b-galactosidase (and two other enzymes,
galatoside permease and thiogalactoside
transacetylase)
13.8 Both the synthesis and degradation of the
certain important enzymes are tightly controlled.
For example, in cell cycle control and in cancer.
Either overexpression or overproduction of a
protein or a failure to turn off and destruct a
protein can lead to cancer.