Enzymes

Definition

• An enzyme is a biological catalyst

• The vast majority of enzymes are proteins,
although a few are RNA based (ribozymes)
• Catalysts (including enzymes) accelerate the
rate of a reaction, but do not affect free
energy changes
Enzymes

• Catalysts (including enzymes) are returned to

their starting state at the end of the reaction
 Some Properties of Enzymes

• Specificity
– Examples: trypsin, chymotrypsin
• Enzyme activities are regulated
• Coenzymes/cofactors: Non-amino acid
components are necessary for the activity of
some enzymes.
Enzymes

Apoenzyme = enzyme without coenzyme

Holoenzyme = enzyme with coenzyme
 Relationship Between Kinetics and
Thermodynamics
A ↔ T* ↔ B
• An enzyme lowers the free
energy of activation for the
reaction. This is another way
of saying that it increases the
reaction velocity.
• An enzyme does not affect
the overall free energy
change of the reaction (and
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therefore, does not change

the equilibrium).
• An enzyme accelerates the
approach to equilibrium.
 Enzymes stabilize the transition state

• An important way that enzymes accelerate reactions

Enzymes

is by stabilizing the transition state

• Enzymes may also provide specific reactive groups
(such as general acids & bases) and they may
transiently form covalent complexes with substrates
 What influences rates of enzyme
reactions?
• Substrate concentration
influences the reaction
rate
• Typically get hyperbolic
curves when plotting
reaction rate versus
substrate concentration
– evidence of
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saturation of enzyme
by substrate
 What influences rates of enzyme
reactions?
• Temperature
– Higher temperatures
usually accelerate reactions
– Too high a temperature will
denature the enzyme
• pH
– pH can influence reaction
Enzymes

rate, ionization of active site

and enzyme stability
– Most enzymes have a
characteristic pH optimum
 Michaelis-Menten Equation
Model:
S = substrate
E = enzyme
P = product
k1, k-1 and k2 are rate constants

Km = Michaelis constant (k-1+k2)/k1

v0 = initial reaction velocity
Enzymes

Vmax = maximum velocity (k2Etot)

[S] = substrate concentration

The Michaelis-Menten
Equation
 Michaelis-Menten Equation

Some implicit assumptions:

1. [S] >> [E]
This means that [S]tot ≈ [S]free
2. Steady-state
Enzymes

i.e., [ES] does not change with time

3. Initial velocity
Before substrate has been depleted, and rate of
back-reaction is negligible
 Michaelis-Menten Equation

• Describes a rectangular
hyperbola when velocity is
plotted as a function of
substrate concentration
• Km is the substrate
concentration giving half-
Enzymes

maximal reaction velocity

• Km is expressed in units of
concentration (e.g., mM, μM)
 Michaelis-Menten Equation

• Km describes the affinity of

enzyme for substrate
• Small Km means high affinity
for substrate
Enzymes

• Large Km means low affinity

for substrate
 kcat (Turnover Number)
• kcat is a measure of the intrinsic
catalytic activity of an enzyme
• The units of kcat are inverse time
(sec-1)
Enzymes
Enzymes
Some Turnover Numbers
 A Linear Plot: Lineweaver-Burk

• Also called double

reciprocal plot
• When 1/v0 is plotted
against 1/[S], it
describes a straight
line with a slope of
(Km/Vmax) and a y-
intercept of 1/Vmax
• Useful for visualizing
Enzymes

effects of enzyme
inhibitors
 Enzyme Inhibition
• Definition: An inhibitor is a compound that
interacts with an enzyme to slow the rate of
an enzyme-catalyzed reaction.
• Reversible inhibitors bind to enzymes by
noncovalent interactions. Enzyme activity
recovers when inhibitor is removed.
• Irreversible inhibitors (inactivators) react
Enzymes

with enzymes through covalent bonds.

Enzyme activity does not recover when
inhibitor is removed.
 Reversible Inhibitors: Competitive
• Inhibitor binds reversibly into the active site
• Inhibitor & substrate therefore compete with each
other for access to the enzyme
Enzymes
 Competitive Inhibitors
• High substrate
concentrations will
reverse the inhibition
• Therefore, competitive
inhibitors change the
apparent Km, but do not
affect kcat.
• Statins are examples of
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drugs that function as

competitive enzyme
inhibitors.
 Reversible Inhibitors: Noncompetitive
• Inhibitor binds reversibly to the enzyme, but at a
different site than the substrate binding site
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 Noncompetitive Inhibitors
• Noncompetitive
inhibitors typically bind
to a site on the enzyme
distinct from the active
site
• These inhibitors bind to
both E and ES
• They reduce the kcat but
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do not affect Km
 Irreversible Inhibitors
• These could more accurately
be called enzyme inactivators
• They covalently modify the
enzyme, destroying its activity
permanently
• Example: aspirin
Aspirin
(acetylsalicylate) transfers its (acetylsalicylate)
acetyl group to a serine
Enzymes

residue in the active site of

cyclooxygenase, permanently
inactivating the enzyme
 Regulation of Enzyme Activity
• Substrate concentration may
regulate enzyme activity, as long
as the [S] is not >> Km
• Covalent modification – often
by phosphorylation – can
modulate the activity of some
enzymes. Examples:
– glycogen phosphorylase (↑)
– glycogen synthetase (↓)
Enzymes

• Rate of enzyme biosynthesis

• Allosteric regulation
 Allosteric Regulation of
Enzyme Activity
• Allosteric literally means
“another site”
• Allosteric regulators bind
outside the active site and
change the enzyme’s activity
(positively or negatively)
• Such modulators can change
K0.5, kcat, or both
Enzymes

• Allosteric enzymes often

deviate from Michaelis-Menten
kinetics
 Allosteric Regulation of Enzyme
Activity
• Allosteric modulators can be:
– Homotropic effectors
(typically the substrate
itself)
– Heterotropic effectors
(often a downstream
metabolite that feeds back
to regulate the initial key
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step in a pathway)
 Enzymes in Clinical Diagnosis
• Cytoplasmic enzymes are normally absent
from the blood (or present in only tiny
amounts)
• Cell damage can cause leakage of these
enzymes, raising their blood levels
• The presence of tissue-specific isoenzymes
(isozymes) in the blood can help diagnose
damage to specific organs.
Enzymes

• Enzymes are favored targets of such tests

because of their specificity and sensitivity