Chapter 6 Enzyme catalysis

1
Chapter 6 Enzyme catalysis

• ? Study history,
• ? General features,
• ? Classification,
• ? Chemical nature,
• ? Kinetics
• ? Mechanism,
• ? Regulation

This will be lectured by Professor Zengyi Chang
on April 21and 28 in the year 2007.
2
The study history of enzymes
Enzymes Biological catalysts that promote and
speed up chemical reactions without themselves
being altered (consumed) in the process. They
determine the patterns of transformations for
chemicals, as well as forms of energy in the
living organisms.
3
The discovery of enzymes as the biocatalysts (1)

• Both enzymology and biochemistry were evolved
  from the 19th century investigation on the nature
  of animal digestion and fermentation.
• Biochemical reactions could not be reproduced in
  the lab initially and was thought (e.g., Louis
  Pasteur) to occur by the action of a vital
  force.
• The idea of catalytic force or contact
  substance promoting fermentation was introduced
  in about 1830s.
• Addition of alcohol to an aqueous extract of malt
  (geminating barley) and saliva precipitated a
  flocculent (??) material which liquefied starch
  paste and converted it into sugar, this material
  was named diastase (1833) (later amylase????).

4
The discovery of enzymes as the biocatalysts (2)

• Pepsin was discovered as the active principle in
  the acid extract of gastric mucosa causing the
  dissolution of coagulated egg white (1834).
• Other soluble ferments discovered in the 19th
  century include trypsin (1857), invertin (later
  invertase and sucrase, 1864), papain (vegetable
  trypsin, 1879), etc.
• Enzyme (something in yeast) was first coined
  for such unorganized ferments by Kühne in 1876.
  
• Enzymes for alcoholic fermentation were found to
  be active in cell free extracts of yeast (1897,
  Eduard Buchner) fermentation is a chemical
  process, not a vital process.

5
The discovery of enzymes as the biocatalysts (3)

• Relationship of initial velocity (V0) and
  substrate concentration (S) was examined.
• A mathematical description was established for
  the kinetics of enzyme action (Michaelis and
  Menten, 1913).
• Weak-bonding interactions between the enzymes and
  their substrates were proposed to distort the
  substrate and catalyze a reaction (Haldane,
  1930s).

Before it was known that enzymes are proteins!!!
John Burdon Sanderson Haldane (1892-1964)

Leonor Michaelis (1875-1949)
Maud Menten (1879-1960)
A British Geneticist
A German
A Canadian
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Enzyme specificity was revealed by studying sugar
conversion (Emil Fischer, 1890S)

• Sugars of known structure were synthesized and
  used as substrates of enzymes.
• The a-methylglucoside was found to be hydrolyzed
  by invertin, but not by emulsin, whereas the
  b-methylglucoside was cleaved by emulsin, but not
  by invertin the enzyme and the glucoside was
  considered to fit (complement) each other like a
  lock and a key.
• Formation of an ES complex was proposed (1894).

8
Enzymes were found to be proteins

• The question of homogeneity of the enzyme
  preparations frustrated the field of enzymology
  for many decades.
• Nitrogen content analysis and various color tests
  (for proteins) led to contradictory results.
• Filterable coenzymes (co-ferments) were
  discovered in Buchners zymase (Harden and Young,
  1906).
• Enzymes were thought to be small reactive
  molecules adsorbed on inactive colloidal
  material, including proteins ( as by R.
  Willstätter in the 1920s).
• Urease (1926, Sumner) and pepsin (1930, Northrop)
  were crystallized and found to be solely made of
  proteins.

9
Urease crystals ( X 728)Sumner, J. B. (1926)
The isolation and crystallization of the enzyme
urease J. Biol. Chem. 69435-441.
10
Pepsin crystals (X90) Northrop, J. H.
(1930) Crystallin pepsin, 1 Isolation and
tests of purity J. Gen . Physiol. 13739-766.
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Not all enzymes are proteins Some RNA molecules
(ribozymes) were found to be catalytic (Sidney
Altman and Thomas Cech, 1981).Ribozymes are
found to promote RNA processing.
Sidney Altman visiting PKU
13
Formation of an enzyme-substrate (ES) complex was
suggested

• The activity of invertase in the presence of
  sucrose survives a temperature that completely
  destroys it if the sucrose is not present (C
  OSullivan and F. W. Tompson, 1890).
• Emil Fishers study on the specifity of invertase
  (1894).
• The rate of fermentation of sucrose in the
  presence of yeast seemed to be independent of the
  amount of sucrose present, but on the amount of
  the enzyme (A. J. Brown, 1902).
• The kinetics of enzyme action was originally
  studied using invertase (a hyperbola when V0 was
  plotted against S).
• The enzyme (E) was thus assumed to form a
  complex. (ES) with the substrate (S) before the
  catalysis.

14
The kinetics of the enzyme-catalyzed reaction
were found to be rather different from those of a
typical chemical reaction
The rate is proportional to the concentration of
the reactant in a typical chemical reaction.
Enzymes however showed a saturation kinetics
formation of ES complex was hypothesized (1902).
15
The enzyme theory of life was formulated

• Enzymes are central to every biochemical process
  (Hofmeister, 1901) life is short and thus has
  to be catalyzed.
• Isolation, purification and physico-chemical
  characterization of enzymes would be important
  for understanding the nature of life.
• Without catalysis, the chemical reactions needed
  to sustain life could not occur on a useful time
  scale.
• Self replication and catalysis are believed to be
  the two fundamental conditions for life to be
  evolved. (RNA is thus proposed to be the type of
  life molecules first evolved).

16
The current understanding on the general features
of enzymes
Extraordinarily powerful Highly specific Be
often regulated.
17
Enzymes are the most remarkable and specialized
biological catalysts

• An enzyme catalyzes a chemical reaction at a
  specifically structured active site, being often
  within the confine of a pocket on the enzyme.
• Enzymes have extraordinary catalytic power, often
  far greater than those non-biological catalysts.
• Enzymes often have a high degree of specificity
  for their substrates.
• Enzymes are often regulated.
• Enzymes usually work under very mild conditions
  of temperature and pH.
• The substance acted on by an enzyme is called a
  substrate, which binds to the active site of an
  enzyme in a complementary manner.

18
Rate enhancement
2 H2O2 ? 2 H2O O2
Fe3 ?1000 fold Hemoglobin ? 1 ,000,000
fold Catalase ? 1 ,000,000,000 fold
200,000 catalytic events/second/subunit (near
the diffusion-controlled limit). The reaction is
sped up by a billion fold!
(a prosthetic group)
Active site
(tetramers)
19
Each enzyme has at least one active site
20
The study of enzymes has immense practical
importance

• Many genetic diseases are caused by a deficiency
  or a total absence of one or more enzymes (e.g.
  Phenylketonuria-PKU, severe-combined
  immunodeficiency-SCID).
• Measurements of the activities of enzymes (in the
  blood or tissues) are important in diagnosing
  certain diseases (e.g., transaminases and liver
  damages).
• Enzymes are important drug targets (aspirin
  inhibits a cyclo-oxygenase of prostaglandin
  biosynthesis).
• Enzymes are widely used in the chemical industry
  and food processing.

21
Many enzymes need non-protein cofactors to help
in catalysis
Cosubstrates?

• The cofactors can be inorganic ions or coenzymes
  (complex organic or metallo-organic molecules).
• Some cofactors bind to the enzyme protein very
  tightly (non-covalently or covalently), they are
  thus called prosthetic groups.
• Only the combination of an apoenzyme with its
  cofactor (i.e., a holoenzyme) is operative (a
  holoenzyme also refers to the assembled form of a
  multiple subunit protein).
• Coenzymes usually function as transient carriers
  of specific function groups.
• Vitamins (organic nutrients required in small
  amount in the diet) have been found to often act
  as precursors of coenzymes.

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(Vitamins)
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Many enzymes have been named by adding the suffix
-ase to the name of their substrate or to a
word/phrase describing their activity

• Urease (hydrolysis of urea).
• Transaminase (transfer amino group from one
  molecule to another).
• RNA polymerase (formation of RNA by
  polymerization).
• But many enzymes are named before this rule was
  established (e.g., pepsin, trypsin).

25
Enzyme classification Enzymes are grouped into
six classes according to the type of reactions
catalyzed.
26
Transfer electrons (hydride ions or H
atoms) play a major role in energy metabolism.
Lactate dehydrogenase
e.g., the transfer of a phosphoryl group from
ATP to many different acceptors.
NMP kinase
Chymotrypsin
the transfer of functional groups to water.
These are direct bond breaking reactions without
being attacked by another reactant such as H2O.
Fumarase
Triose phosphate isomerase
In chemical terms, they would be described
as elimination and addition reactions.
Leading to the formation of C-C, C-S, C-O, C-N
bonds.
Aminoacyl-tRNA synthetase
27
Each enzyme is given a systematic name and a
unique 4-digit identification number for
identification by the Enzyme Commission (E.C.) of
IUBMB (since 1964)
lactate NAD pyruvate NADH H
Lactate dehydrogenase (lactateNAD
oxidoreductase)
1
Indicates type of cofactor
Indicates type of substrate
28
The chemical nature of enzyme catalysis
29
A chemical reaction is studied in two aspects
thermodynamics and kinetics

• Chemical thermodynamics whether a chemical
  process can occur at all (spontaneously
  /exergonic or not ?G values).
• Chemical kinetics deals with the rate of a
  chemical process (V values).
• Many thermodynamically feasible reactions do not
  actually occur in the universe (chaos is thus
  avoided!).
• The universe as we know it is as much controlled
  by the laws of chemical kinetics as by the laws
  of chemical thermodynamics.

30
The directionality and extent of a reversible
reaction is determined by the free energy
difference of the reactants and the products

• A reversible reaction like will reach a
  equilibrium state at a constant temperature,
  where the ratio of reactant and product
  concentrations remain unchanged over time, such a
  ratio was designated as the equilibrium constant
  by Guldberg and Waage (1864) .
• The ' is for biochemical
  reactions, which is determined at pH 7.0.
• The value of K 'eq is determined by the standard
  free energy change of the reaction.

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for
DG
For each 10-fold change in K ' eq,the ?G'o
changes only by 1.36 kcal mol -1 (or 5.69 kJ
mol-1)
o
32
The rate (velocity) of a chemical reaction
reflects how fast the reactants disappear (or
products appear)

• For S ?P, the reaction rate is the amount of S
  that reacts per unit time V k S.
• The rate constant k is first order for a single
  substrate reaction (having units of reciprocal
  time, e.g., s-1) and is second order for a double
  substrate reaction (units can be M-1s-1).

33
The rate constant of a chemical reaction is
determined by the value of its activation energy
(?G ) according to the transition state theory

• It was proposed that reacting molecule or
  molecules must pass through a high energy
  transition state on the way to become the
  product(s).
• The transition state is not a chemical species of
  any significant stability, but a fleeting
  molecular moment in which bonds are both broken
  and formed.
• Transition State theory Enzymes catalyze
  chemical reactions by stabilizing their
  transition states.

34
The rate of a reaction is determined by the
value of activation energy (?G )
A lower activation energy means a faster
reaction rate.
kT
k ? e -
?G
/ RT
The relationship between k and ?G is inverse and
exponential!
h
35
An enzyme provides an alternative pathway for the
conversion of the substrates to the products.
36
Enzymes make the rate constants larger and only
catalyze reactions that are thermodynamically
favorable.
An enzyme provides an alternative pathway for the
conversion of the substrates to the products,
thus lowers the activation energy and speeds up
the reaction.
37
The kinetics of enzyme catalysis Steady state
kineticsPre-steady state kinetics
38
A hyperbolic curve between V0 and S was
revealed by in vitro studies using purified
enzymes

• It was the initial velocity (rate), V0, that was
  measured, so the change of S could be ignored
  (S , being generally five or six orders of
  magnitude higher than E, can be regarded as
  constant).
• The catalysis was assumed to occur as
• The enzyme will become saturated at high S the
  V0 will not be affected by S at high S.

39
Vmax is extrapolated from the plot V0
approaches but never quite reaches Vmax.
The effect on V0 of varying S is measured when
the enzyme concentration is held constant.
Hyperbolic relationship between V0 and
S (similar to the O2 binding curve of myoglobin)
40
A mathematical relationship between V0 and S
was established ( Michaelis and Menten, 1913
Briggs and Haldane, 1925)
k 1
k 2

• E S ES E P
• Formation of ES is fast and reversible.
• The reverse reaction from P?S (k-2 step) was
  assumed to be negligible.
• The breakdown of ES to product and free enzyme is
  the rate limiting step for the overall reaction.
• ES was assumed to be at a steady state its
  concentration remains constant over time.
• Thus V0 k2ES

(
)
41
E S ES E P
k 1
k 2
k -1
Km is called the Michaelis constant.

• Steady-state assumption
• Rate of ES formationrate of ES breakdown
• k1(Et-ES)Sk-1ES k2ES
• (Et is the total enzyme concentration.)
• Solve the equation for ES

V0 k2ES
42
The maximum velocity is achieved when all the
enzyme is saturated by substrate, i.e., when
ES Et. Thus Vmax
k2Et
The Michaelis-Menten Equation
43
When S gtgt Km
When S ltlt Km
The Michaelis-Menten Equation nicely describes
the experimental observations.
The substrate concentration at which V0 is half
maximal is Km
44
The Vmax and Km values of a certain enzyme can be
measured by the double reciprocal plot (i.e.,
the Lineweaver-Burk plot).
45
The double reciprocal plot 1/V0 vs 1/S
46
The Michaelis-Menten equation, but not their
approximated mechanism applies to a great many
enzymes

• Most enzymes (except the regulatory enzymes) have
  been found to follow the Michaelis-Menten
  kinetics, but their actual mechanisms are usually
  more complicated (by having more intermediate
  steps) than the one assumed by Michaelis and
  menten.
• The values of Vmax and Km alone provide little
  information about the number, rates, or chemical
  nature of discrete steps in the reaction.

47
The actual meaning of Km depends on the reaction
mechanism
k 1

• For
• If k2 is rate-limiting, k2ltltk-1,
  Km k-1/k 1
• Km equals to the dissociation constant (Kd) of
  the ES complex
• Km represent a measure of affinity of the enzyme
  for its substrate in the ES complex.
• If k2gtgtk-1, then Km k2/k1.
• If k2 and k-1 are comparable, Km is a complex
  function of all three rate constants.

k -1
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Vmax is determined by kcat, the rate constant of
the rate-limiting step

• Vmax kcatEt
• kcat equals to k2 or k3 or a complex function of
  both, depending on which is the rate-limiting
  step.
• kcat is also called the turnover number the
  number of substrate molecules converted to
  product in a given unit of time per enzyme
  molecule when the enzyme is saturated with
  substrate.

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40,000,000 molecules of H2O2 are converted to H2O
and O2 by one catalase molecule within one second!
51
The kinetic parameters kcat and Km are often
studied and compared for different enzymes

• Km often reflects the normal substrate
  concentration present in vivo for a certain
  enzyme.
• The catalytic efficiency of different enzymes is
  often compared by comparing their kcat/Km ratios
  (the specificity constant).
• when
• SltltKm
• kcat/Km is an apparent second-order rate constant
  (with units of M-1S-1), relating the reaction
  rate to the concentrations of free enzyme and
  substrate.

52
The value of kcat/Km has an upper limit (for the
perfected enzymes)

• It can be no greater than k1.
• The decomposition of ES to E P can occur no
  more frequently that E and S come together to
  form ES.
• The most efficient enzymes have kcat/Km values
  near the diffusion-controlled limit of 108 to 109
  M-1S-1.

53
Catalytic perfection (rate of reaction being
diffusion-controlled) can be achieved by a
combination of different values of kcat and Km.
54
Rate enhancement is often used to describe the
efficiency of an enzyme
kcat
catalyzed
Rate enhancement ratio of the rates of the
catalyzed and the uncatalyzed reactions.
uncatalyzed
kcat
55
Rate enhancement by selected enzymes
Uncatalyzed rate (kun, s-1)
Catalyzed rate (kcat, s-1)
Rate enhancement (kcat/kun)
Nonenzymatic half-life
Enzyme
56
Enzyme-catalyzed reactions of two or more
substrates can also be analyzed by the
Michaelis-Menten approach

• Each substrate will have one characteristic Km
  value.
• Noncovalent ternary complex (with two substrates
  bound to the enzyme concurrently) may or may not
  be formed for the bisubstrate reactions depending
  on the mechanism.
• Steady-state kinetics can often help distinguish
  these two mechanisms.

57
In those enzyme-catalyzed bisubstrate reactions
where a ternary complex is formed, the two
substrates may either bind in a random sequence
or in a specific order.
58
For those reactions where ternary complex is
formed
Maintaining the concentration of one substrate
(S2) constant, the double reciprocal plots made
by varying the concentration of the other
substrate (S1) will intersect.
59
No ternary complex is formed in the Ping-Pong (or
double displacement) mechanism The first
substrate is converted to a product that leaves
the enzyme active site before the second
substrate enters.
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For enzymes having Ping-Pong mechanisms (ternary
complex not formed).
Maintaining the concentration of one substrate
(S2) constant, the double reciprocal plots made
by varying the concentration of the other
substrate (S1) will not intersect.
As S2 increases, Vmax increases, as does the Km
for S1.
S1
61
Rates of individual steps for an enzyme-catalyzed
reaction may be obtained by pre-steady state
kinetics

• The enzyme (of large amount) is used in substrate
  quantities and the events on the enzyme are
  directly observed.
• Rates of many reaction steps may be measured
  independently.
• Very rapid mixing and sampling techniques are
  required (the enzyme and substrate have to be
  brought together in milliseconds and measurements
  also be made within short period of time).

62
Rapid kinetics or pre-steady- state
kineticsis applied to the observation of rates
of systems that occur in very short time
intervals (usually ms or sub-ms scale ) and
very low product concentrations. This period
covers the time from the enzyme encountering
its target (either a substrate, inhibitor or
some other ligands) to the point of system
settling to equilibrium.
The concentration of ES will rise from zero to
its steady-state value.
(ms or sub-ms)
63
Solutions are forced together very rapidly.
Stopped-flow apparatus for pre-steady state
kinetics (since 1940s)
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Quench flow apparatus for rapid kinetics
65
Enzyme catalysis can be slowed or halted by
specific inhibitors.Such inhibitors are
important pharmaceutical agents and useful in
understanding the action mechanism of enzymes.
66
Competitive inhibitors have structures similar to
the substrates and thus inhibit enzyme catalysis
by binding to the active site in a reversible way
E P
ES
E
KI
EI
How would the Km and Vmax be affected?
X
67
The presence of competitive inhibitors alters the
Km but not the Vmax of enzymes

Apparent Vmax and Km values Vmax unchanged, Km
increases
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Uncompetitive inhibitors binds at a site distinct
from the substrate binding site the inhibitor
binds only to the ES complex
How would Km and Vmax be affected?
E P
???????
E
ES
Uncompetitive inhibitors are present only for
enzymes catalyzing reactions of two or more
substrates (with ordered substrate binding)
analogs of S2 will act as uncompetitive inhibitor
for the enzyme (relative to S1)
ESI
X
69
The presence of uncompetitive inhibitors alter
both the Km and the Vmax of an enzyme
Both Vmax and Km decreases (but Vmax/Km
unchanged).
70
A mixed inhibitor also binds at a site distinct
from the substrate binding site, but binds to
either E or ES
Noncompetitive inhibitor binding of I does not
affect binding of S Vmax decreases, Km unchanged.
E P
E
ES
EI
ESI
Mixed inhibitors are present for enzymes of
random ordered substrate binding.
X
71
The presence of mixed inhibitors alter both the
Km and the Vmax of an enzyme
Vmax decreases, Km increases.
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73
Irreversible inhibitors chemically modify or form
tight noncovalent interactions with functional
groups in the active site of enzymes.A suicide
inhibitor (or mechanism-based inactivator) is
converted into an irreversible inhibitor by the
action of the specific enzyme.
74
Enzymes catalyze chemical reactions via both
noncovalent and covalent interactions

• Noncovalent interactions between substrates and
  enzymes will generate a binding energy (?GB),
  which will lower the activation energy of the
  reactions.
• Transient chemical reactions (i.e., covalent
  interactions) often occur between substrates and
  functional groups in the active sites of enzymes,
  thus providing an alternative reaction path.

75
Weak interactions between enzyme and substrate
are optimized in the transition state

• Enzyme and substrate was proposed to complement
  each other like a lock and the key (Emil
  Fischer, 1894).
• Years later, it was realized that an enzyme
  completely complementary to its substrate would
  be a very poor enzyme!
• According to the transition state theory, an
  enzyme must be complementary to the reaction
  transition state of the reactant (Haldane, 1930
  Pauling, 1946).

76
An effective enzyme must have its active
site complementary to the transition state of the
reaction.
Activation energy increases!
ES
E-transition state
E P
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The transition state theory of enzyme catalysis
has strong supporting evidences

• The idea of transition-state analogs was
  suggested in accordance with the transition state
  theory (Pauling, 1940s) and were later proved to
  be correct such analogs bind to enzymes 102 to
  106 times more tightly than normal substrates.
• The idea of catalytic antibodies was also
  suggested by this theory (Jencks, 1969) and was
  proved to be correct later (Lerner and Schultz,
  1980s).

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Transition-state
Transition-state analogs can be designed
according to the proposed reaction mechanism and
used as antigens for making catalytic antibodies.
Catalytic antibodies
Transition-state analog
Transition-state
Catalytic antibodies
Transition-state analog
79
The binding energy made available by the
noncovalent enzyme-substrate interactions often
provide a major driving force for enzyme
catalysis.
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Binding energy can be used for selecting specific
substrates and overcome the ?G

• The reduction in entropy of oriented substrates.
• Desolvation of the substrates.
• Distortion of substrates for converting to the
  transition state.
• Proper alignment of catalytic function groups via
  induced fit (conformational change) in the enzyme
  active site.
• The consumption of binding energy in such
  processes will help lower the ?G , thus
  increasing the reaction rate.

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Specific catalytic groups in the enzyme active
site act via transient covalent interactions.
82
Many chemical reactions can be promoted
by general acid-base catalysis
Temporarily donating or accepting a proton.
83
Side chains of many amino acid residues can act
as general acid-base
84
Each enzymes usually act at an optimal pH value
or range
pH optimun at around 7.8
pH optimum at around 1.6
85
The catalytic mechanism of chymotrypsin a member
of the serine protease family catalyzes the
hydrolytic cleavage of peptide bonds adjacent to
aromatic amino acid residues (with a rate
enhancement of at least 109).
Principles illustrated Transition-state
stabilization General acid-base
catalysis Covalent catalysis.
86
The catalytically important groups of
chymotrypsin were identified by chemical labeling
studies

• Organic fluorophosphates such as
  diisopropylphosphofluoridate (DIPF) irreversibly
  inactivate chymotrypsin (and other serine
  proteases) and reacts only with Ser195 (out of
  the 25 Ser residues).

87
A second catalytically important residue, His57,
was discovered by affinity labeling with
tosyl-L-phenylalanine chloromethylketone (TPCK)
TPCK alkylates His 57 Inactivation can be
inhibited by b-phenylpropionate (competitive
inhibitor) TPCK modification does not occur
when chymotrypsin is denatured in urea.
88
Rapid initial burst kinetics indicates an
acyl-enzyme intermediate

• The kinetics of chymotrypsin is worked out by
  using artificial substrates (esters), yielding
  spectroscopic signals upon cleavage to allow
  monitoring the rate of reactions.

Colorless substrate
Yellow product
Fast
Slow
This reaction is far slower than the hydrolysis
of peptides!
Km 20 mM Kcat 77 s-1
89
The catalysis of chymotrypsin is biphasic as
revealed by pre-steady state kinetics
Slow phase (enzymes will be able to act again
only after a slow deacylation step)
burst (fast) phase (rapid acylation of all
Enzymes leading to release of p-nitrophenol)
Milliseconds after mixing
90
Determination of the crystal structure of
chymotrypsin (1967) revealed a catalytic triad
Ser195, His57, Asp102.
91
Chymotrypsin three polypeptide chains linked by
multiple disulfide bonds a catalytic triad.
Active site
His57
Ser195
Asp102
Cleft for binding extended substrates
Trypsin, sharing a 40 identity
with chymotrypsin, has a very similar structure.
92
A catalytic triad has been found in all serine
proteases the Ser is thus converted into a
potent nucleophile (subtilisin has no homology
with other Ser protease members, but has the
triad)
93
The Peptide Bond has partial (40) double bond
character as a result of resonance of electrons
between the O and N
The hydrolysis of a peptide bond at neutral pH
without catalysis will take 10-1000 years!
94
Chymotrypsin (and other serine proteases) acts
via a mixture of covalent and general acid-base
catalysis to cleave (not a direct attack of water
on the peptide bond!)
95
Formation of the ES complex
Asp102 functions only to orient His57.
E
S
Formation of ES1
ES1
The peptide bond to be cleaved is positioned by
the binding of the side chain of an adjacent
hydrophobic residue in a special hydrophobic
pocket.
96
His57 acts as a general base in deprotonating
Ser195, the alkoxide ion then acts as a
nucleophile, attacking the carbonyl carbon.
ES1
Ser195 forms a covalent bond with the peptide
(acylation) to be cleaved. a trigonal C is
turned into a tetrahedral C. The tetrahedral
oxyanion intermediate is stabilized by the NHs
of Gly193 and Ser195
Pre-acylation
Preferential binding of the transition state
oxyanion hole stabilization of the negatively
charged tetrahedral intermediate of the
transition state.
oxyanion hole
97
His57 acts as a general acid in cleaving the
peptide bond.
ES1
Acylation
Releasing of P1
The amine product is then released from
the active site with the formation of an
acyl-enzyme covalent intermediate.
Acyl-E
98
Entering of S2
Acyl-E
ES2
Water (the second substrate) then enters the
active site.
99
His57 acts as a general base again, allowing
water to attack the acyl-enzyme
intermediate, forming another tetrahedral oxyanion
intermediate, again stabilized by the NHs of
Gly193 and Ser195 (similar to step 2)
Pre-deacylation
ES2
100
EP2
His57 acts as a general acid again in breaking
the covalent bond between the enzyme and
substrate (deacylation) (similar to Step 3).
Deacylation
101
Recovered enzyme
Release of P2
E
The second product (an acid) is released from
the active site, with the enzyme recovered to
its original state.
EP2
102
1st substrate
2nd product
E
EP2
ES
The proposed complete catalytic cycle of
chymotrypsin (rate enhancement 109) A Ping-Pong
Mechanism
1st product
ES2
Acyl-E
Acylation phase
Deacylation phase
2nd substrate
103
The specificity of serine proteases is determined
by the structural features of a substrate binding
pocket
Val Val
104
A dynamic process for chymotrypsin catalysis A
Ping Pang mechanism.
Importance of the residues was exmined
by site-directed mutagenesis The Ser and His
residues are far more important than the Asp
residue!
105
The activity of many enzymes (and proteins) are
regulated (modulated), allowing them to function
at the proper time and place in the living
organisms.
106
Groups of enzymes need to work together in a
sequential pathway to carry out a given metabolic
process (e.g., the biosythesis of Ile from Thr)
Feedback inhibition (1961) Ile was found to
specifically and reversibly inhibit the first
enzyme in the pathway.
Ile is not a steric analogue of the substrate of
Thr dehydratase!
107
The activities of enzymes can be regulated via
mainly three principle ways

• Allosteric regulation (noncovalent modifications,
  reversible)
• Covalent modifications (reversible)
• Proteolytic cleavage (irreversible).
• (Gene regulation changing the amount of specific
  enzymes).

108
Allosteric regulation The binding of allosteric
modulators (small signal molecules, often small
metabolites or cofactors) at specific sites
(allosteric sites) distinct from the active site
triggers conformational changes that are
transmitted to the active site (intramolecular
signal transduction).
109
Allosteric modulators can be either inhibitory or
stimulatory
110
Allosteric enzymes are often oligomeric The
aspartate transcarbamoylase (ATCase) consists of
two catalytic trimers and three regulatory dimers
catalytic trimer
regulatory dimer
regulatory dimer
regulatory dimer
regulatory dimer
catalytic trimer
regulatory dimer
catalytic trimer
regulatory dimer
111
The conformational change seems to follow the
Concerted (MWC) model
CTP
CTP
CTP
CTP
T- state
Major conformational differences were observed
for the ATCase strucutures in the presence of CTP
and ATP.
ATP
ATP
ATP
ATP
The active site is 50 A away from the
allosteric site.
R-state
112
The kinetic properties of allosteric enzymes
diverge from Michaelis-Menten behavior
Some allosteric enzymes exhibit sigmoidal
saturation curves, i.e., the active sites show
cooperativity the binding of substrate to one
active site favors the conversion of the entire
enzyme into the R state. such regulation is
called homotrophic (substrates are
modulators). Heterotrophic M differs from S.
113
But the free catalytic trimers (when separated
from the regulatory dimers) of aspartate
transcarbamoylase does exhibit Michaelis-Menten
kinetics.
114
Activities of some enzymes are regulated
(activated or inhibited) via reversible covalent
modificationswith the addition and removal
often catalyzed by separate enzymes.
115
Glycogen phosphorylase and many others (for 1/3
to ½ of all eukaryotic proteins)
Protein kinases
Protein phosphatases
Glutamine synthetase
Dinitrogenase reductase RNA polymerase
116
Sites of modification can be far away from the
active sites
Phosphorylation sites
Active sites
Glycogen phosphorylase a
117
Phosphorylation affects the structure, the
thermodynamics and the kinetics of enzymes.
118
Some enzymes (and other proteins) are activated
via proteolytic cleavage of precursor proteins
(zymogens or preproteins).Many proteases
activated this way can be inactivated by
inhibitor proteins tightly-bound in the active
sites.
119
Active chymotrypsin and trypsin are produced from
inactive zymogens via proteolytic cleavage, with
conformational changes exposing the active
sites.
120
Some regulatory enzymes use multiple regulatory
mechanisms

• The bacterial Gln synthetase is regulated via
• Allostery
• Reversible covalent modification
• A regulatory protein.

121
The complex, highly regulated symphony of the
life phenomena depend much on the complex
regulation of various key enzymes.
122
Summary

• Enzymes are powerful and highly specific
  biocatalysts with the activities of many of which
  highly regulated.
• Vitamins were found to act as precursors of the
  non-protein cofactors of enzymes.
• The thousands of enzymes have been grouped into
  six classes.
• Enzymes only speed up the reaction rates and do
  not affect the reaction equilibria.
• Many enzymes follow the Michaelis-Menten kinetics.

123
Summary

• Pre-steady state kinetics is often more
  informative than steady state kinetics.
• Inhibitors are useful as tools in enzyme study or
  as drugs in pharmacology.
• Enzymes catalyze reactions via both noncovalent
  and covalent interactions.
• Enzyme catalysis is believed to occur by
  stabilizing the transition state of a specific
  reaction.
• The action mechanisms of serine proteases are
  among the best studied.

124
Summary

• The activities of many enzymes are regulated via
  allostery, reversible covalent modifications
  (especially phosphorylation) or proteolytic
  cleavage.
• The study of enzymes is valuable in understanding
  the nature of life, as well as in medical and
  industrial practices.

125
See you all at Biochemistry II Good luck for the
final exam!