高等生化學

Advanced Biochemistry

Enzymes

陳威戎
Preface
One way in which this condition might be fulfilled would be if
the molecules when combined with the enzyme, lay slightly
further apart than their equilibrium distance when [covalently
joined], but nearer than their equilibrium distance when
free….Using Fischer’s lock and key simile, the key does not fit
the lock quite perfectly but exercises a certain strain on it.
- J. B. S. Haldane, Enzymes, 1930

Catalysis can be described formally in terms of a stabilization

of the transition state through tight binding to the catalyst.
- William P. Jencks, article in Advances in Enzymology, 1975
Two fundamental conditions for life

1. The living entity must be able to self-replicate.

2. The organism must be able to catalyze chemical
reactions efficiently and selectively.

~ Without catalysis, chemical reactions could not

occur on a useful time scale, and thus could not
sustain life.
Extraordinary catalytic power of enzymes

1. High degree of specificity for their substrates

2. Accelerate chemical reactions tremendously
3. Function in aqueous solutions under very mild
conditions of temperature and pH
Enzymes
1. An Introduction to Enzymes
2. How Enzymes Work
3. Enzyme Kinetics as an Approach to
Understanding Mechanism
4. Examples of Enzymatic Reactions
5. Regulatory Enzymes
 An Introduction to Enyzmes

1. Late 1700s- digestion of meat by secretions of the stomach.

2. 1800s- conversion of starch to sugar by saliva and plant extracts.
3. Louis Pasteur (1850s)- fermentation of sugar into alcohol by
yeast is catalyzed by “ferments”.
~ ferments are inseparable from living yeast cells- “vitalism”
4. Eduard Buchner (1897)- yeast extracts could ferment sugar to
alcohol.
~ fermentation was promoted by molecules that
continue to function when removed from cells.
5. Frederick W. Kühne- called these molecules “enzymes”.
 An Introduction to Enyzmes

6. James Sumner (1926)- isolation and crystallization of

urease. ~ postulated that “all enzymes are proteins”
7. John Northrop and Moses Kunitz (1930s)- crystallized
pepsin, trypsin, and other digestive enzymes.
~ all found to be proteins
8. J. B. S. Haldane (1850s)- wrote “Enzymes”
~ weak bonding interactions between an enzyme and
its substrate might be used to catalyze a reaction.
9. Late 20th century- purification and elucidation of the
structure and mechanism of many enzymes.
 1. Most enzymes are proteins

1. With the exception of a small group of catalytic RNA and DNA

molecules, all enzymes are proteins.
2. The catalytic activity depends on the integrity of their native
protein conformation.
3. Enzymes have MWs ranging from 12,000 to more than 1 million.
4. Some enzymes require no chemical groups for activity, others
require an additional chemical component called a “cofactor”
(inorganic ions) or a “coenzyme” (complex organic or
metalloorganic molecules).
Prosthetic group
Holoenzyme vs. Apoenzyme
2. Enzymes are classified by the reactions they catalyze

Many enzymes have been named:

1. By adding the suffix “-ase” to their substrate ex: urease
2. For a broad function before the specific reaction catalyzed
was known ex: pepsin
3. For their sources ex: trypsin
4. By the Nomenclature Committee of IUBMB
ex: ATP:glucose phosphotransferase (E.C. 2.7.1.1.)
Enzymes
1. An Introduction to Enzymes
2. How Enzymes Work
3. Enzyme Kinetics as an Approach to
Understanding Mechanism
4. Examples of Enzymatic Reactions
5. Regulatory Enzymes
Binding of a substrate to an enzyme at the active site
 How Enzymes Work
1. Enzymes affect reaction rates, not equilibria.
2. Reaction rates and equilibria have precise thermodynamic
definitions.
3. A few principles explain the catalytic power and specificity
of enzymes.
4. Weak interactions between enzyme and substrate are
optimized in the transition state.
5. Binding energy contributes to reaction specificity and
catalysis.
6. Specific catalytic groups contribute to catalysis.
1. Enzymes affect reaction rates, not equilibria

: Standard free energy change

: Biochemical standard free-energy change
: Activation energy
Ground state: Substrate (S) ; Product (P)
Transition state
Reaction intermediates (ES or EP)
Catalysts enhance reaction rates by lowering
activation energies.
Reaction coordinate diagram for a chemical reaction
Reaction coordinate diagram comparing enzyme-
catalyzed and uncatalyzed reactions
 2. Reaction rates and equilibria have precise
thermodynamic definitions
For an equilibrium such as
Equilibrium constant: Keq ;
The relationship between K’eq and G’o:
Reaction rate is determined by [reactants] and rate constant: k
For a unimolecular reaction S→P, the rate (or velocity), V is
expressed by a rate equation: (first-order reaction)
For a bimolecular reaction, (second-order reaction)
 3. A few principles explain the catalytic power
and specificity of enzymes

Enzymes are extraordinary catalysts:

1. The rate enhancements that the enzymes bring about are in
the range of 5 to 17 orders of magnitude.
2. Enzymes are very specific.

The source of energy for the lowering of activation energy:

1. The rearrangements of covalent bonds during an enzyme-
catalyzed reaction.
2. The noncovalent interactions between enzyme and substrate.
 3. A few principles explain the catalytic power
and specificity of enzymes

: binding energy ; derived from enzyme-substrate interaction.

A major source of free energy used by enzymes to lower the
activation energies of reactions.

How enzymes use noncovalent binding energy?

1. Catalytic power of enzymes is derived from the free energy
released in forming many weak interactions with their substrates.
This binding energy contributes to specificity and to catalysis.
2. Weak interactions are optimized in the reaction transition state.
Enzyme active sites are complementary not to the substrates but
to the transition states.
 4. Weak interactions between enzyme and substrate
are optimized in the transition state

1. Emil Fischer (1894) proposed that enzymes were structurally

complementary to their substrates, so that they fit together like a
lock and key. (An elegant but misleading idea!)
2. Michael Polani (1921), Haldane (1930), and Linus Pauling (1946)
proposed the modern notion of enzymatic catalysis: in order to
catalyze reactions, an enzyme must be complementary to the
reaction transition state.
Weak binding interactions between the enzyme and the substrate
provide a substantial driving force for enzymatic catalysis.
Complementary shapes of a substrate and its binding site on
an enzyme

The enzyme dihydrofolate reductase with its substrate NADP+

unbound (left) and bound (right).
An imaginary enzyme (stickase) designed to catalyze
breakage of a metal stick
5. Binding energy contributes to reaction specificity
and catalysis

Prominent physical and thermodynamic factors contributing to

1. A reduction in entropy

2. The solvation shell of hydrogen-bonded water

3. The distortion of substrates

4. The need for proper alignment of catalytic functional groups

“Induced fit”, a mechanism postulated by Daniel Koshland (1958)

Role of binding energy in catalysis
Rate enhancement by entropy reduction
6. Specific catalytic groups contribute to catalysis

1. General acid-base catalysis

Proton transfers mediated by molecules other than water.
Amino acid side-chains in the active site of an enzyme can act
as proton donors or acceptors.
2. Covalent catalysis
A transient covalent bond is formed between the enzyme and
the substrate.
Amino acid side-chains and functional groups of coenzymes
can serve as nucleophiles in the formation of covalent bonds.
3. Metal ion catalysis
Ionic interactions between enzyme-bound metal and substrate.
Mediate oxidation-reduction reactions.
How a catalyst circumvents
unfavorable charge
development during cleavage
of an amide
Amino acids in general acid-base catalysis
Covalent and general acid-base catalysis
Enzymes
1. An Introduction to Enzymes
2. How Enzymes Work
3. Enzyme Kinetics as an Approach to
Understanding Mechanism
4. Examples of Enzymatic Reactions
5. Regulatory Enzymes
 Enzyme Kinetics as an Approach to
Understanding Mechanism
1. Substrate concentration affects the rate of enzyme-
catalyzed reactions.
2. The relationship between substrate concentration and
reaction rate can be expressed quantitatively.
3. Kinetic parameters are used to compare enzyme activities.
4. Many enzymes catalyze reactions with two or more
substrates.
5. Pre-steady state kinetics can provide evidence for specific
reaction steps.
6. Enzymes are subject to reversible or irreversible inhibition.
7. Enzyme activity depends on pH.
1. Substrate concentration affects the rate of enzyme-
catalyzed reactions- Initial rate (velocity)

One simplifying approach in enzyme kinetics experiments is to

measure the initial rate (or initial velocity), designated V0,
when [S] is much greater than the concentration of enzyme, [E].
At relatively low concentrations of substrate, V0 increases almost
linearly with an increase in [S].
A point is reached beyond which increases in V0 are vanishingly
small as [S] increases. This plateau-like region is close to the
maximum velocity, Vmax.
1. Substrate concentration affects the rate of enzyme-
catalyzed reactions- ES complex

Victor Henri (1903) proposed that the combination of an enzyme

with its substrate to form an ES complex is a necessary step in
enzymatic catalysis.
Leonor Michaelis and Maud Menten (1913) postulated that the
enzyme first combines reversibly with its substrate to form an
ES complex in a relatively fast reversible step:

The ES complex then breaks down in a slower second step to

yield the free enzyme and the reaction product P:
1. Substrate concentration affects the rate of enzyme-
catalyzed reactions- Steady state

The enzyme is first mixed with a large excess of substrate, there is

an initial period, the pre-steady state, during which [ES] builds
up.
The reaction quickly achieves a steady state in which [ES]
remains approximately constant over time.
The concept of a steady state was introduced by G. E. Briggs and
Haldane in 1925. Analysis of these initial rates is referred to as
steady-state kinetics.
Effect of substrate concentration on the initial
velocity of an enzyme-catalyzed reaction
2. The relationship between substrate concentration
and reaction rate can be expressed quantitatively

[Et]: total enzyme concentration

[Et]-[ES]: free or unbound enzyme
Since [S] is far greater than [Et], the amount of substrate bound
by the enzyme is negligible compared with the total [S].
With these conditions in mind, the following steps lead us to an
expression for V0 in terms of easily measurable parameters.
2. The relationship between substrate concentration
and reaction rate can be expressed quantitatively

The modern derivation of Michaelis-Menten equation:

Step 1: the formation and breakdown of ES complex

Step 2: Steady-state assumption: the rate of formtion of

ES is equal to the rate of its breakdown
2. The relationship between substrate concentration
and reaction rate can be expressed quantitatively
Step 3: Solve the equation for ES
Km=
(Michaelis-Menten constant)

Step 4: Express Vo in terms of ES

Vmax occurs when enzyme is saturated ([ES]=[Et])
Vmax can be defined as k2[Et]
Dependence of initial velocity on substrate concentration
3. Kinetic parameters are used to compare enzyme
activities
All enzymes that exhibit a hyperbolic dependence of V0 on [S] are
said to follow Michaelis-Menten kinetics.
When k2 is rate-limiting, k2<<k-1 and Km reduces to k-1/k1, which is
defined as the dissociation constant, Kd, of the ES complex.
Km represents the affinity of the enzyme for its substrate.
Define a more general rate constant, kcat = Vmax/[Et], also called
“turnover number”

Specificity constant, kcat/Km: the best way to compare the

catalytic efficiencies of different enzymes or substrates.
Transformation of the Michaelis-Menten equation:
The double-reciprocal (Lineweaver-Burk) plot
Versions useful in the determination of Km and Vmax.
4. Many enzymes catalyze reactions with two or
more substrates
 5. Pre-steady state kinetics can provide evidence
for specific reaction steps

A ternary complex is formed in A Ping-Pong (double-displacement)

the reaction steps. pathway.
6. Enzymes are subject to reversible or irreversible
inhibition
Enzyme inhibitors are molecular agents that interfere with
catalysis, slowing or halting enzymatic reactions.
There are two broad classes of enzyme inhibition:
1. Reversible inhibition
(1) Competitive inhibition
(2) Uncompetitive inhibition
(3) Mixed inhibition (a special case: Noncompetitive inhibition)
2. Irreversible inhibition
Suicide inactivators (mechanism-based inactivators)
Three types of reversible inhibition- Competitive inhibition
Three types of reversible inhibition- Uncompetitive inhibition
Three types of reversible inhibition- Mixed inhibition
 Irreversible inhibition

Irreversible inhibitors bind

covalently with or destroy
functional groups on an enzyme
essential for activity or form a
particularly stable noncovalent
interaction.
Suicide inactivators: unreactive
until they bind to the active site of
an specific enzyme, also called
“mechanism-based inactivators”.-
suitable for “rational drug design”
7. Enzyme activity depends on pH

The pH-activity profiles of two enzymes

Enzymes
1. An Introduction to Enzymes
2. How Enzymes Work
3. Enzyme Kinetics as an Approach to
Understanding Mechanism
4. Examples of Enzymatic Reactions
5. Regulatory Enzymes
Examples of Enzymatic Reactions

1. The chymotrypsin mechanism involves acylation

and deacylation of a serine residue.
2. Hexokinase undergoes induced fit on substrate
binding.
3. The enolase reaction mechanism requires metal
ions.
4. Lysozyme uses two successive nucleophilic
displacement reactions.
1. The chymotrypsin mechanism involves
acylation and deacylation of a serine residue

Bovine pancreatic chymotrypsin: a protease that catalyze the

hydrolytic cleavage of peptide bonds.
Specific for peptide bonds adjacent to aromatic amino acids.
General acid-base catalysis and covalent catalysis.
Enhance the rate of peptide bond hydrolysis by at least 109-fold.
The reaction has two phases: (1) acylation phase ; (2)
deacylation phase.
Structure of chymotrypsin
Structure of chymotrypsin
Pre-steady state kinetic evidence for an acyl-enzyme intermediate
The pH dependence of chymotrypsin-catalyzed reactions
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 1
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 2
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 3
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 4
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 5
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 6
Hydrolytic cleavage of a peptide bond by chymotrypsin- Step 7
Some common nucleophiles and
electrophiles in biochemistry
2. Hexokinase undergoes induced fit on substrate binding

Yeast hexokinase (Mr 107,862) is a bisubstrate enzyme that

catalyzes the following reversible reaction:

Hexokinase favors the reaction with glucose by a factor of 106.

Reaction rates increase greatly in the presence of the functional
phosphoryl group acceptor (glucose).
General acid-base catalysis and transition-state stabilization.
 Induced fit in hexokinase

Hexokinase has a U-shaped structure. The ends pinch toward each other
in a conformational change
induced by binding of D-glucose.
3. The enolase reaction mechanism requires metal ions

A glycolytic enzyme, enolase, catalyze the reversible dehydration

of 2-phosphoglycerase to phosphoenolpyruvate:

Yeast enolase (Mr 93,316) is a dimer with 436 amino acid

residues per subunit.
Metal ion catalysis, general acid-base catalysis and transition-
state stabilization.
 Two-step reaction catalyzed by enolase

The carboxyl group of 2-PGA is coordinated by two Mg2+ at the active site.
A proton is abstracted in step 1 by general base catalysis (Lys345), and the
resulting enolic intermediate is stabilized by the two Mg2+ ions.
Elimination of the -OH in step 2 is facilitated by general acid catalysis (Glu211).
The substrate, 2-PGA in the enolase active site
Structure-activity correlations: Chymotrypsin-catalyzed amide
hydrolysis
Transition-state analogs: Catalytic antibodies
4. Lysozyme uses two successive nucleophilic
displacement reactions

A natural antibacterial agent found in tears and egg whites.

Hen egg white lysozyme (Mr 14,296) is a monomer with 129 a.a.
The structure revealed four disulfide bonds and a cleft containing
the active site.
Substrate: peptidoglycan in bacterial cell walls.
Cleaves the b1→4 glycosidic bond between two types of sugar
residues in a molecule: N-acetylmuramic acid (Mur2Ac, NAM)
and N-acetylglucosamine (GlcNAc, NAG)
Key catalytic amino acid residues : Glu35 and Asp52
Ribbon diagram of the hen egg white lysozyme
Reaction catalyzed by hen egg white lysozyme
Two proposed pathways of lysozyme reaction
Two proposed pathways of lysozyme reaction
Two proposed pathways of lysozyme reaction
Two proposed pathways of lysozyme reaction
Ribbon diagram of the covalent lysozyme-substrate intermediate
Enzymes
1. An Introduction to Enzymes
2. How Enzymes Work
3. Enzyme Kinetics as an Approach to
Understanding Mechanism
4. Examples of Enzymatic Reactions
5. Regulatory Enzymes
Regulatory enzymes are modulated in a variety of ways

1. Allosteric enzymes function through reversible

noncovalent binding of regulatory compounds
called allosteric modulators or allosteric effectors.

2. Other enzymes are regulated by reversible covalent

modification.
Regulatory Enzymes
1. Allosteric enzymes undergo conformational changes in
response to modulator binding.
2. In many pathways a regulated step is catalyzed by an allosteric
enzyme.
3. The kinetic properties of allosteric enzymes diverge from
Michaelis-Menten behavior.
4. Some regulatory enzymes undergo reversible covalent
modification.
5. Phosphoryl groups affect the structure and catalytic activity of
proteins.
6. Multiple phosphorylations allow exquisite regulatory control.
7. Some enzymes and other proteins are regulated by proteolytic
cleavage of an enzyme precursor.
1. Allosteric enzymes undergo conformational changes
in response to modulator binding
1. Allosteric enzymes are significantly different from those of
simple nonregulatory enzymes. (Some are structural.)
2. Allosteric enzymes generally have one or more regulatory, or
allosteric, sites for binding the modulator.
3. Allosteric enzymes are generally larger and more complex than
nonallosteric enzymes. Most have two or more subunits.
4. Aspartate transcarbamoylase (ATCase), which catalyzes an
early reaction in the biosynthesis of pyrimidine nucleotides,
has 12 polypeptide chains organized into catalytic and
regulatory subunits.
Subunit interactions in an allosteric enzyme, and
interactions with inhibitors and activators
Two views of the regulatory enzyme
Aspartate transcarbamoylase
2. In many pathways a
regulated step is
catalyzed by an
allosteric enzyme

Feedback inhibition
Heterotropic allosteric inhibition
3. The kinetic properties of allosteric enzymes diverge from
Michaelis-Menten behavior- Sigmoid curve (homotropic)

Not Km!
3. The kinetic properties of allosteric enzymes diverge from
Michaelis-Menten behavior- Effects of modulators
3. The kinetic properties of allosteric enzymes diverge from
Michaelis-Menten behavior- Less common type of modulation
4. Some regulatory enzymes undergo reversible
covalent modification
1. Phosphorylation:
1/3 to 1/2 of all proteins in a eukaryotic cell are
phosphorylated
2. Adenylylation:
3. Uridylylation:
4. Methylation:
Methyl-accepting chemotaxis protein of bacteria
5. ADP-ribosylation:
Dinitrogenase reductase of bacteria
Some enzyme modification reactions- Phosphorylation
Some enzyme modification reactions- Adenylylation
Some enzyme modification reactions- Uridylylation
Some enzyme modification reactions- ADP-ribosylation
Some enzyme modification reactions- Methylation
5. Phosphoryl groups affect the structure and
catalytic activity of proteins

1. The attachment of phosphoryl groups is catalyzed by protein

kinases; removal by protein phosphatases
2. Addition of a phosphoryl group to a Ser, Thr, or Tyr introduces
a bulky, charged group into a moderately polar region.
3. Oxygen atoms can hydrogen-bond with:
amide group of peptide backbone at the start of an a-helix
charged guanidinium group of Arg
4. Two negative charges can repel neighboring Asp or Glu
Regulation of glycogen phosphorylase activity by
covalent modification
6. Multiple phosphorylation allow exquisite regulatory control
Multiple regulatory phosphorylations
7. Some enzymes and other proteins are regulated
by proteolytic cleavage of an enzyme precursor