ABS 311

Chapter 6:
Enzymes the Catalysts of Life
Enzyme Catalysis

 Many reactions that are energetically

favorable do not occur without a catalyst, …
why?
 The activation energy is an energy barrier
that must be overcome before the reaction
can occur
 The transition state is a high energy
intermediate form that must be achieved by
the reactant(s)
Enzyme Catalysis
Enzyme Catalysis
 Molecules in the metastable state are
thermodynamically unstable but do not
possess enough energy to exceed the
activation energy barrier
 A catalyst lowers the activation energy, thus
enhancing the rate of reaction, without being
consumed in the reaction
Enzymes as Biological Catalysts

 Three functions of enzymes:

 Increase the rate of reactions by lowering the
activation energy
 Bind substrates in a manner that facilitates
interactions and stabilizes the transition state
 Enhance rates only, the reaction must already be
thermodynamically favorable (exergonic)
Enzymes as Biological Catalysts
 Enzymes were identified as protein
catalysts in 1926 with the crystallization
of urease (ended belief in vitalism)

 In the 1980’s another class of enzymes,

ribozymes, were discovered. These are
RNA molecules possessing catalytic
capabilities
Enzymes as Biological Catalysts
 The active site of an enzyme is where the
substrate binds and catalysis occurs
 Amino acids which make up the active site are
generally distant from each other in the
primary sequence of the protein
 Substrate binding frequently triggers a
conformational change
 The active site has high specificity for the
substrate
Enzymes as Biological Catalysts
Enzymes as Biological Catalysts

 Even though only a few amino acids are

involved in the active site, the overall
tertiary structure is critical to enzyme
function
 The amino acids found at an enzyme
active site are typically: cysteine,
histidine, serine, aspartate, glutamate or
lysine
Enzymes as Biological Catalysts
Enzymes as Biological Catalysts
 Some enzymes require a prosthetic
group for catalytic activity. These
are not part of the polypeptide
chain(s), but are post-translationally
bound via covalent or non-covalent
bonds. Egs., heme, metal ions
(apoenzyme + prosthetic group =
holoenzyme) -
OOC CH2CH2 CH2CH2COO
-

H3C CH3
N N
Fe(II)
H2C N N
CH CH3

CH3 CH CH2
Enzymes as Biological Catalysts
 Substrate specificity refers to the highly
selective nature of binding at the active
site, often discriminating between two
very similar molecules
 Group specificity allows for more
general enzyme binding to related
substrates, (eg. carboxypeptidase,
hexokinase)
IUB Enzyme Commission
Classification
 Enzymes are grouped in six major
classes based on their functions;
oxidoreductases, transferases,
hydrolases, lyases, isomerases, and
ligases
 Each EC name has four parts, eg.
EC 3.4.17.1 is carboxypeptidase
indicating its; major class, subclass,
sub-subclass and serial number
IUB Enzyme Commission
Classification
Enzyme Catalysis
 Enzymes are sensitive to temperature and pH
deviations
 Human enzymes exhibit maximum efficiency at
37oC
 Organisms such as plants, protists and bacterium
must function at the temperature of their
environment
 Enzymes only function at a narrow pH range due
to the pH dependence of active site functional
groups (or substrate functional groups)
Enzyme Catalysis

The effect of
temperature and
pH on the
reaction rate of
enzyme-
catalyzed
reactions
Enzyme Binding Site
 Enzyme catalyzed reactions proceed at
rates 107 to 1014 times faster than
uncatalyzed reactions…..why? How?
 Consider uncatalyzed reactions:
 Collisions must occur with correct orientation
 Substrate must overcome highly unfavorable
transition state
 Enzyme Binding Site
 The Induced Fit
Model of
substrate binding
assumes that
binding at the
active site distorts
both the enzyme
and the substrate
 Enzyme Binding Site
 Substrate activation
can occur via:
 Conformational change
inducing bond
distortion
 Enzyme may
donate/accept protons
(acid/base catalysis)
 Enzyme may
donate/accept
electrons
Enzyme Catalysis
 After a catalytic event occurs the active site
releases to its original state
 Thousands of catalytic events can occur at
an active site per second
Enzyme Binding Site
Enzyme Kinetics
 Enzyme kinetics is the quantitative
assessment of enzyme turnover rates
 Attention is focused on initial reaction rates
where the velocity is linear due to constant
turnover.
 In a Michaelis-Menton plot initial velocities are
plotted vs. substrate concentration, notice that
vi increases linearly with increasing [S] until
the enzyme approaches saturation
Enzyme Kinetics

 Michaelis-
Menton Kinetics:
The relationship
between
reaction velocity
and substrate
concentration
 Enzyme Kinetics

 In MM kinetics the
equation: (inset in plot)
 Ef + S = ES = Ef + P
shows the formation of the
transient enzyme-substrate
complex
 From the MM model the
MM equation for vi was
derived quantifying the
relationship between
velocity and substrate
concentration
Enzyme Kinetics
 Km – Michaelis
constant, indicator of
substrate binding
affinity
 Vmax – maximum
obtainable initial
velocity at high
substrate
concentrations
Enzyme Kinetics
 Consider Vmax and Km conditions:
 #1 Low substrate concentration [S]<<Km
 MM eq becomes v = Vmax[S]/Km
 First-order region of MM plot where reaction
increases linearly with increasing S
 #2 High substrate concentration [S]>>Km
 MM eq becomes v = Vmax[S]/[S] = Vmax
 Velocity is independent of [S] variation and
essentially constant. Zeroeth-order region of MM
plot, due to saturating substrate concentrations
Enzyme Kinetics
 #3 [S] = Km
 At this point v = Vmax/2 which is the
definitive point called the Michaelis
constant.

 Turnover number, kcat, rate at which

substrate molecules are converted to
product by a single enzyme molecule
when the enzyme is operating at its
maximum velocity
 kcat = Vmax/[Et]
Enzyme Kinetics
Enzyme Kinetics

 The Lineweaver-Burk
Double-Reciprocal Plot
Enzyme Inhibitors at the
Molecular Level
 Enzyme inhibitors are substances that
interfere with enzyme function and can be
classified as reversible or irreversible
 Irreversible – binds covalently to enzyme causing
an irrevocable loss of catalytic activity
 Reversible – binds non-covalently interferring with
enzyme turnover
 Competitive – binds at active site
 Non-competitive – binds somewhere other than active
site
Enzyme Inhibitors
Enzyme Inhibitors at the
Cellular Level
 Enzyme rates must be continuously adjusted
to keep them tuned to the needs of the cell

 Regulation that depends on interactions of

substrates and products with an enzyme is
called substrate-level regulation

 Increases in substrate levels result in

increased reaction rates, whereas increased
product levels lead to lower rates
Enzyme Inhibitors at the
Cellular Level
 Cells can turn enzymes on and off as needed by
two mechanisms: allosteric regulation and
covalent modification

 Usually enzymes regulated this way catalyze the

first step of a multi-step sequence

 By regulating the first step of a process, cells are

able to regulate the entire process
Enzyme Regulation

Allosteric
Regulation of
Enzyme Activity
Feedback Inhibition
 It is not in the best interests of a cell for
enzymatic reactions to proceed at the maximum
rate

 In feedback (or end-product) inhibition, the

final product of an enzyme pathway negatively
regulates an earlier step in the pathway


Enzyme Regulation
 Substrate-level regulation is an important
form of cellular enzymatic control
 Allosteric regulation – (“other site”) a molecule
other than the substrate binds at another site and
signals enzyme to slow down or speed up
 Feedback inhibition – a product of a metabolic
pathway signals the enzyme at the beginning of
the pathway
 Allosteric effector molecules may decrease or
increase enzyme activity, and therefore are known
as allosteric inhibitors or allosteric activators
Allosteric Enzyme Regulation
Covalent Enzyme Regulation
 Many enzymes are subject to covalent
modification

 Activity is regulated by addition or removal of

groups, such as phosphate, methyl, acetyl
groups, etc.
Covalent Modification
 The reversible addition of phosphate groups is a
common covalent modification
 Phosphorylation occurs most commonly by transfer of a
phosphate group from ATP to the hydroxyl group of Ser, Thr, or
Tyr residues in a protein
 Protein kinases catalyze the phosphorylation of other proteins

 Dephosphorylation, the removal of phosphate groups from

proteins, is catalyzed by protein phosphatases

 Depending on the enzyme, phosphorylation may be associated

with activation or inhibition of the enzyme
Covalent Modification
 The activation of a protein by a one-time,
irreversible removal of part of the polypeptide
chain is called proteolytic cleavage

 Proteolytic enzymes of the pancreas, trypsin,

chymotrypsin, and carboxypeptidase, are
examples of enzymes synthesized in inactive
form (as zymogens) and activated by
cleavage as needed
Enzyme Regulation
– Covalent Modification
Regulation via phosphorylation Regulation via proteolytic cleavage
RNA Molecules as Enzymes:
Ribozymes
 Some RNA molecules have been found to have
catalytic activity; these are called ribozymes

 Self-splicing rRNA from Tetrahymena

thermophila and ribonuclease P are examples

 It is thought by some that RNA catalysts predate

protein catalysts, and even DNA
Chapter 6 - Homework
 6-1, 6-2, 6-4, 6-5, 6-6, 6-7, 6-9, 6-10, 6-11