FUNDAMENTALS OF

BIOCHEMISTRY

Fourth Edition
Donald Voet - Judith G. Voet - Charlotte W. Pratt

Chapter 11:
Enzyme Catalysis
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Enzyme Catalysis
Enzyme activity and classification
Enzyme specificity and cofactors
Transition-State Diagrams
Catalytic Mechanisms
Lysozyme
Serine Proteases
2

Enzymes
Enzymes differ from ordinary chemical catalyst in several important
respects:
Higher reactions rates: The rates of enzymatically catalyzed
reactions are typically several of orders of magnitude greater than
those of the corresponding uncatalyzed reactions.
Milder reactions conditions: Enzymatically catalyzed reactions occur
under relative mild conditions (below 100C, atmospheric pressure,
and nearly neutral pH)
Greater reaction specificity
Capacity for regulation: The catalytic activities of many enzymes
vary in response to the concentrations of substances other than
their substrates.

Enzymes

Reaction Coordinate Diagrams

E + S ES E + P

reactants (substrate, S)
products (P)
enzyme (E)
transition states
G
-

spontaneous rxn

activation energy

Reaction Coordinate Diagrams

Catalysts act by providing a reaction

pathway with a transition state whose
free energy is lower than in the
uncatalyzed reaction.
lowers the activation energy
does not change Grxn

S
E + S ES E + P

Reaction Coordinate Diagrams

Chemical reactions commonly consist of
several steps. For a two-step reaction such
as:
A I P
there are two transition sates and two
activation energy barriers.
The step with the highest transition state
acts as a bottleneck and is said to be the
rate-limiting step.

Enzyme Classification
Enzymes are commonly named by
appending the suffix -ase to the name
of the enzyme's substrate or to a
phrase describing the enzyme's
catalytic action.
urease catalyzes the hydrolysis of
urea
alcohol dehydrogenase catalyzes
the oxidation of alcohols by
removing hydrogen.
There are six major classes of
enzymatic reactions.
know these
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Enzyme Specificity
Substrate-binding site consists of an indentation
or cleft on the surface of an enzyme molecule
that is complementary in shape to the substrate
(geometric complementarity).
Amino acids residues that form the binding site
are arranged to specifically attract the substrate
(electronic complementarity).
Enzymes act on specific substrates via:
van der Waals
electrostatic
hydrogen bonding
hydrophobic interaction
Nearly all enzymes that participate in chiral
reactions are absolutely stereospecific.
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Enzyme Specificity
Enzymes vary in the degree of geometric specificity. A few enzymes are
absolutely specific for only one compound.
Most enzymes, however, catalyze the reactions of a small range of
related compounds, but with different efficiencies.
Alcohol dehydrogenase catalyzes the oxidation of ethanol to
acetaldehyde faster than it oxidizes methanol to formaldehyde (or
isopropanol to acetone).
Digestive enzymes

10

Cofactors
Cofactors may be:
metal ions (Cu2+, Fe3+, Zn2+)
organic molecules (coenzymes)

cosubstrates: NAD+, NADP+

prosthetic groups are permanently associated with their protein

(heme group in myoglobin, hemoglobin and cytochromes)

A catalytically active enzyme-cofactor complex is called a holoenzyme.

An inactive protein resulting from the removal of a holoenzymes
cofactor is called an apoenzyme.
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How do Enzymes Work?

Catalysis can occur through proximity and orientation effects. By simply
binding their substrates, enzymes facilitate their catalyzed reactions in 4
ways:
Enzymes bring substrates into contact with the catalytic groups.
Enzymes bind their substrates in the proper orientation.
Charged groups may help stabilize the transition state of the
reaction (electrostatic catalysis).
Enzymes freeze out the relative translational and rotational motions
of their substrates and catalytic groups.

12

How do Enzymes Work?

An enzyme may bind the transition state
of the reaction it catalyzes with greater
affinity that its substrate or products.

enzymes that preferentially bind the

transition state structure increase its
concentration and therefore
increase the reaction rate.
-

rationale for drug design and

enzyme inhibitors

13

Catalytic Mechanisms
The types of catalytic mechanisms that enzymes employ have been
classified as:
Acid-base catalysis
Metal ion catalysis
Covalent catalysis

Proximity and orientation effects

Preferential binding of the transition state complex

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Acid-Base Catalysis
General acid catalysis is a process
in which proton transfer from an
acid lowers the free energy of a
reactions transition state.
hydrolysis of peptide bonds
phosphate group reactions

The side chains of Asp, Glu, His,

Cys, Tyr, and Lys have pKs in or
near physiological pH range, which
permits them to act as acid and/or
base catalysts.

activity is sensitive to pH
15

Effects of pH on Activity
Most enzymes are active within only a narrow pH
range (5-9).
binding of substrate to enzyme
the ionization states of the amino acid residues
the ionization of the substrate
the variation of protein structure

Inflection point on the curve often provides valuable

clues to the identities of the amino acid residues
essential for enzymatic activity.
pK of ~ 4: Asp or Glu
pK of ~6: His
pK of ~10: Lys

16

RNase A
RNase A is an example of
enzymatically mediated acidbase catalysis.
secreted by the pancreas
into the small intestine
hydrolyzes RNA to its
component nucleotides
120 amino acids
pI = 9.6

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His 12 (acting as a base) abstracts a proton from

an RNA 2-OH group.
This promotes its nucleophilic attack on the
adjacent phosphorous atom.
His 119 acts as an acid.
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After leaving group departs, water enters the

active site, and the 2,3-cyclic intermediate is
hydrolyzed.
H12 now acts as an acid and H119 as a base.
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Metal Ion Catalysis

Nearly one-third of all known enzymes require metal ions for catalytic
activity.
metalloenzymes contain tightly bound metal ions (Fe2+, Fe3+, Cu2+,
Mn2+, Co2+)
Na+, K+, Ca2+ play a structural role rather than a catalytic role
Mg2+ and Zn2+ may be either structural or catalytic
Metal ion cofactors:
bind to substrates to orient them properly
mediate oxidation-reduction reactions
electrostatically stabilize or shield negative charges

20

Metal Ion Catalysis

Carbonic anhydrase:

catalyzes the rapid conversion of:

CO2 + H2O HCO3- + H+

contains an essential Zn2+ ion (prosthetic

group) tetrahedrally coordinated by three
His side chains, and 1 H2O molecule
Zn2+ ion polarizes a water molecule to
form OH-, which nucleophilically attacks
the substrate CO2 to yield HCO3-.

21

Covalent Catalysis
Covalent catalysis accelerates reaction rates through the transient
formation of a catalyst-substrate covalent bond.
serine proteases: acyl-serine intermediate
protein kinases and phosphatases: phospho-amino acid interm.

nucleophilic catalysis: covalent bond is formed by the reaction of a

nucleophile group on the catalyst with an electrophilic group on the
substrate.
-

side chains of His, Cys, Asp, Lys and Ser can participate in
covalent catalysis by acting as nucleophiles.

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Covalent Catalysis
The nucleophilicity of a substance is closely related to its basicity.
biological nucleophiles are negatively charged or contain unshared
electron pairs
biological electrophiles include positively charged groups, contain
unfilled valence electron shell, or contain an electronegative atom.

23

Lysozyme
Lysozyme is an enzyme that destroys bacteria cell
walls.
hydrolyzing the (14) glycosidic linkage from
NAM to NAG in cell wall peptidoglycans.
occurs widely in the cells of vertebrates

probably functions as bactericidial agent

example of covalent catalysis and acid-base

catalysis.

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Bacterial Cell Wall

25

Lysozyme
Lysozyme is an enzyme that destroys
bacteria cell walls.
HEW lysozyme in complex with
NAG
Glu 35 and Asp 52 catalyze the
hydrolysis of the glycosidic
linkage

Asp negative charge side

chain functions to
electronically stabilize the
intermediate

Glu acts as an acid catalyst

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27

Serine Proteases
Serine proteases are a group of proteolytic enzymes, which include
digestive enzymes and specialized proteins that participate in
development, blood clotting, inflammation, etc.

Serine in their active site

Digestive Enzymes:

synthesized by the pancreas and secreted into the small intestine.

catalyze the hydrolysis of peptide bonds (with different specificities)
chymotrypsin: specific for a bulky hydrophobic residue (F, Y, W)
trypsin: specific for a positively charged residue (R, K)
elastase: specific for a small neutral residue (A, G, V)

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Identifying Important Amino Acids

Chemical labeling studies helped identify
chymotrypsins catalytically important
groups.
Diisopropylphosphofluoridate (DIPF):
diagnostic test for the presence of the
active site Ser of serine proteases
irreversibly inactivates the enzyme
reacts only with Ser 195 of
chymotrypsin, thereby demonstrating
that this residue is the enzymes active
site Ser.

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Identifying Important Amino Acids

A second catalytically important residue, His 57, was discovered though
affinity labeling.
substrate analog specifically binds at the active site.
tosyl-L-phenylalanine chloromethylketone (TPCK)

30

Serine Proteases
Chymotrypsin, trypsin and elastase are strikingly
similar.
primary structure: ~40% identical
all have a reactive Ser and a catalytically
essential His

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Serine Proteases

32

33

Mechanism of Serine Proteases

34

35

36

37

38

Serine Proteases - Intermediate

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Zymogens
Zymogens are inactive precursors of
proteolytic enzymes.
acute pancreatitis
the newly liberated N-term Ile residue
moves from the surface of the protein
to an internal position, where its free
cationic forms an ion pair with Asp 194.

without this conformation change,

the enzyme cannot properly bind
its substrate or stabilize the
intermediate

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Objective - Chapter 11
Know what properties distinguish enzymes from other catalysts
Know how different enzymes are classified
Be able to explain the factors that influence an enzymes substrate
specificity

Know why certain enzymes require cofactors

Be able to sketch and/or label the various parts of a transition state
diagram for a given reaction (with and without a catalyst)

Know the types of catalytic mechanisms that certain enzymes employ.

Be able to describe them in detail (ex: know how protein functional
groups can act as acid and base catalysts.)

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Objective - Chapter 11
Know the function of RNase A and how it works
Know the function of lysozyme and how it works
Be able to describe how serine proteases work
Know what distinguishes the three types of serine proteases discussed
in class (binding specificity)

Know the ways that active site residues can be identified

Know what zymogens are and why there are advantages of synthesizing
them.

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