What Are the Mechanisms of Enzyme-

Induced Rated Accelerations?

• Mechanisms of catalysis:
•

– Entropy loss in ES formation

– Destabilization of ES
– Covalent catalysis
– General acid/base catalysis
– Metal ion catalysis
– Proximity and orientation

Why Is the Binding Energy of ES Crucial

to Cataylsis?

Competing effects determine the position of

ES on the energy scale
• Try to mentally decompose the binding
effects at the active site into favorable and
unfavorable
• The binding of S to E must be favorable
• But not too favorable!
• Km cannot be "too tight" - goal is to make
the energy barrier between ES and EX‡
small

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What Roles Do Entropy Loss and Destabilization
of the ES Complex Play?

Raising the energy of ES raises the rate

• For a given energy of EX‡, raising the energy

of ES will increase the catalyzed rate
• This is accomplished by
– a) loss of entropy due to formation of ES
– b) destabilization of ES by
• strain
• distortion
• desolvation

Formation of the ES complex results in a loss of entropy. Prior to binding, E and S

are free to undergo translational and rotational motion. By comparison, the ES
complex is a more highly ordered, low-entropy complex.

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 Substrates typically lose waters of hydration in the formation
of the ES complex. Desolvation raises the energy of the ES
complex, making it more reactive.

Electrostatic destabilization of a substrate may arise from juxtaposition of like

charges in the active site. If such charge repulsion is relieved in the course of
the reaction, electrostatic destabilization can result in a rate increase.

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 What Are the Mechanisms of
Catalysis?
Serine Proteases are good examples of
Covalent catalysis
• Enzyme and substrate become linked in a
covalent bond at one or more points in the
reaction pathway
• The formation of the covalent bond provides
chemistry that speeds the reaction

General Acid-base Catalysis

Catalysis in which a proton is transferred in the

transition state
• "Specific" acid-base catalysis involves H+ or
OH- that diffuses into the catalytic center
• "General" acid-base catalysis involves acids
and bases other than H+ and OH-
• These other acids and bases facilitate
transfer of H+ in the transition state

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Catalysis of p-nitrophenylacetate
hydrolysis by imidazole—an example
of general base catalysis. Proton
transfer to imidazole in the transition
state facilitates hydroxyl attack on the
substrate carbonyl carbon.

Liver alcohol dehydrogenase

catalyzes the transfer of a
hydride ion ( H:-) from NADH
to acetaldehyde (CH3CHO),
forming ethanol (CH3CH2OH).
An active-site zinc ion
stabilizes negative charge
development on the oxygen
atom of acetaldehyde, leading
to an induced partial positive
charge on the carbonyl C
atom. Transfer of the
negatively charged hydride ion
to this carbon forms ethanol.

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An example of proximity effects in catalysis. (a) The
imidazole-catalyzed hydrolysis of p-nitrophenylacetate is
slow, but (b) the corresponding intramolecular reaction is
24-fold faster (assuming [imidazole] = 1 M in [a]).

What Can Be Learned from Typical

Enzyme Mechanisms?

• Serine proteases and aspartic proteases are

good examples
• Knowledge of the tertiary structure of an
enzyme is important
• Enzymes are the catalytic machines that sustain
life

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 The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA

• All involve a serine in catalysis - thus the name

• Ser is part of a "catalytic triad" of Ser, His, Asp
• Serine proteases are homologous, but locations
of the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
always as His-57, Asp-102, Ser-195

Comparison of the
amino acid sequences
of chymotrypsinogen,
trypsinogen, and
elastase. Each circle
represents one amino
acid. Numbering is
based on the sequence
of chymotrypsinogen.
Filled circles indicate
residues that are
identical in all three
proteins. Disulfide
bonds are indicated in
yellow. The positions of
the three catalytically
important active-site
residues (His57, Asp102,
and Ser195) are
indicated.

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 Serine Protease Mechanism

A mixture of covalent and general acid-base

catalysis
• Asp-102 functions only to orient His-57
• His-57 acts as a general acid and base
• Ser-195 forms a covalent bond with
peptide to be cleaved
• Covalent bond formation turns a trigonal C
into a tetrahedral C
• The tetrahedral oxyanion intermediate is
stabilized by N-Hs of Gly-193 and Ser-195

The catalytic triad

of chymotrypsin .

8
 The substrate-binding pockets of trypsin, chymotrypsin, and
elastase. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical
Institute. Not to be reproduced without permission. )

Diisopropylfluorophosphate (DIFP) reacts with active-site serine residues of

serine proteases (and esterases), causing permanent inactivation.

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A detailed mechanism for the
chymotrypsin reaction. Note the
low-barrier hydrogen bond
(LBHB) in (c) and (g).

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11
12
13
14
The “ oxyanion
hole” of
chymotrypsin
stabilizes the
tetrahedral
oxyanion
transition states
of the mechanism

What Factors Influence Enzymatic

Activity?

Six points:
• Rate slows as product accumulates
• Rate depends on substrate availability
• Genetic controls - induction and repression
• Enzymes can be modified covalently
• Allosteric effectors may be important
• Zymogens, isozymes and modulator
proteins may play a role

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Enzymes regulated by covalent modification are called interconvertible enzymes. The
enzymes (protein kinase and protein phosphatase, in the example shown here) catalyzing
the conversion of the interconvertible enzyme between its two forms are called converter
enzymes. In this example, the free enzyme form is catalytically active, whereas the
phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertible
enzyme represents an -OH group on a specific amino acid side chain in the protein (for
example, a particular Ser residue) capable of accepting the phosphoryl group.

Proinsulin is an 86-
residue precursor to
insulin (the sequence
shown here is human
proinsulin). Proteolytic
removal of residues 31
to 65 yields insulin.
Residues 1 through 30
(the B chain) remain
linked to residues 66
through 87 (the A chain)
by a pair of interchain
disulfide bridges.

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 The proteolytic activation of chymotrypsinogen.

The cascade of
activation steps leading
to blood clotting. The
intrinsic and extrinsic
pathways converge at
Factor X, and the final
common pathway
involves the activation of
thrombin and its
conversion of fibrinogen
into fibrin, which
aggregates into ordered
filamentous arrays that
become cross-linked to
form the clot.

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The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic
and produces pyruvate from glucose via glycolysis. It needs LDH to regenerate NAD+ from
NADH so glycolysis can continue. The lactate produced is released into the blood. The
muscle LDH isozyme (A4) works best in the NAD+-regenerating direction. Heart tissue is
aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate
to fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited by
excess pyruvate so the fuel won’t be wasted.

What Are the General Features of Allosteric

Regulation?

Action at "another site"

• Enzymes situated at key steps in metabolic
pathways are modulated by allosteric
effectors
• These effectors are usually produced
elsewhere in the pathway
• Effectors may be feed-forward activators or
feedback inhibitors
• Kinetics are sigmoid ("S-shaped")

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 Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot characteristic
of normal Michaelis - Menten-type enzyme kinetics.

Cyclic AMP- dependent protein kinase (also known as PKA) is a 150- to 170-kD R2C2
tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10-8
M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits are
enzymatically active as monomers.

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 Glycogen Phosphorylase
Allosteric Regulation and Covalent
Modification
• Pi is a positive homotropic effector
• ATP is a feedback inhibitor, and a negative
heterotropic effector
• Glucose-6-P is a negative heterotropic
effector (i.e., an inhibitor)
• AMP is a positive heterotropic effector (i.e.,
an activator)

The mechanism of
covalent modification
and allosteric regulation
of glycogen
phosphorylase. The T
states are blue and the
R states blue-green.

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In this diagram of the glycogen phosphorylase
dimer, the phosphorylation site (Ser14) and the
allosteric (AMP) site face the viewer. Access to
the catalytic site is from the opposite side of the
protein. The diagram shows the major
conformational change that occurs in the N-
terminal residues upon phosphorylation of
Ser14. The solid black line shows the
conformation of residues 10 to 23 in the b, or
unphosphorylated, form of glycogen
phosphorylase. The conformational change in
the location of residues 10 to 23 upon
phosphorylation of Ser14 to give the a
(phosphorylated) form of glycogen
phosphorylase is shown in yellow. Note that
these residues move from intrasubunit contacts
into intersubunit contacts at the subunit
interface. [Sites on the two respective subunits
are denoted, with those of the upper subunit
designated by primes (‘).]
(Adapted from Johnson, L. N., and Barford, D., 1993.
The effects of phosphorylation on the structure and
function of proteins. Annual Review of Biophysics and
Biomolecular Structure 22:199-232.)

Regulation of GP by Covalent
Modification
• In 1956, Edwin Krebs and Edmond Fischer
showed that a ‘converting enzyme’ could
convert phosphorylase b to phosphorylase a
• Three years later, Krebs and Fischer show
that this conversion involves covalent
phosphorylation
• This phosphorylation is mediated by an
enzyme cascade

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 The hormone-activated enzymatic cascade that leads to activation of glycogen
phosphorylase.

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