ENZIM
ENZIM
Oleh:
Maria Ulfah, S.Si, M.Pd
JURUSAN PEND.BIOLOGI FPMIPA
IKIP PGRI SEMARANG
What Are Enzymes?
Most enzymes are
Proteins (tertiary
and quaternary
structures)
Act as Catalyst to
accelerates a reaction
Not permanently
changed in the
process
Enzymes
Are specific for
what they will
catalyze
Are Reusable
End in –ase
-Sucrase
-Lactase
-Maltase
Enzyme-Substrate Complex
The substance
(reactant) an
enzyme acts on is
the substrate
Substrate
Joins
Enzyme
Formation of an enzyme-substrate complex
How do enzymes
Work?
Enzymes work
by weakening
bonds which
lowers
activation
energy
Active Site
A restricted region of an enzyme
molecule which binds to the substrate
Active
Site
Substrate
Enzyme
The enzyme active site (features)
The catalytic site is relatively small compared
with the rest of the enzyme. Why are many
enzymes so big then?
The catalytic site is a three-dimensional entity
Substrates are bound to enzymes by multiple
weak, non-covalent interactions (electrostatic
bonds, hydrogen bonds, van der Waals
forces, hydrophobic interactions)
The specificity of binding depends on the
precisely defined arrangement of atoms in
an active site
Emil Fischer (over
100 years ago): came
up with the “lock and
key” hypothesis to
describe enzymesubstrate interactions
Active site of cytochrome P-450
Induced Fit
A change in
the shape of
an enzyme’s
active site
Induced by the
substrate
“Induced fit” model: a
more refined model
that takes into
account the enzyme
assumes a
complimentary shape
to that of its substrate
only after substrate
binds to the enzyme.
More dynamic
scenario compared to
the lock and key
hypothesis
Induced Fit
A change in the configuration of an
enzyme’s active site (H+ and ionic
bonds are involved)
Induced by the substrate
substrate
Active Site
Enzyme
induced fit
What Affects Enzyme Activity?
Three factors:
1. Environmental Conditions
2. Cofactors and Coenzymes
3. Enzyme Inhibitors
1. Environmental Conditions
1. Extreme Temperature are the most
dangerous
- high temps may denature (unfold) the
enzyme.
2. pH (most like 6 - 8 pH near neutral)
3. Ionic concentration (salt ions)
Environmental factors affecting enzyme activity
2. Cofactors and Coenzymes
Inorganic substances (zinc, iron)
and vitamins (respectively) are
sometimes need for proper
enzymatic activity.
activity
Example:
Iron must be present in the
quaternary structure hemoglobin in order for it to
pick up oxygen.
KINETIKA ENZIM
Michaelis-Menten model of
enzyme kinetics (Vmax & Km)
Key element in their model is the
existence of the ES complex
Rate of catalysis (V) increases with
increasing [S], where V is defined as the
number of moles of product formed per
second
When enzyme concentrations are constant, V is
linearly proportional to [S] WHEN [S] IS SMALL.
At high [S] (when S is in vast excess of the
[enzyme]), V is nearly independent of [S]
The Michaelis-Menten equation
Km & Vmax
Km = the Michaelis constant
Defined as the [substrate] at which the
reaction rate is half of its maximal value
Used to define relative affinity of an
enzyme for its substrate
The higher the Km value, the lower the
affinity
Vmax: describes the maximal rate of
product formation when [S] is high
Under such conditions all of the existing
“pool” of enzyme active sites are full
From Vmax an enzyme’s turnover
number can be determined (expressed
as the number of substrate molecules
converted into product per unit time)
Double-reciprocal
(lineweaver-Burk) plot
Used to calculate Km &
Vmax
Also used to characterize
mechanisms of enzyme
inhibition by specific
compounds
Data expressed as 1/V
versus 1/[S]: gives a
straight line
Calculating Km and Vmax
Allosteric enzymes do not conform
to Michaelis-Menten kinetics
Yield a sigmoidal curve on a V versus S
plot (not hyperbolic as seen under
Michaelis-Menten conditions)
Sigmoidal curve indicates cooperative
binding (binding of one molecule of S
affects affinity and binding of
additional S molecules)
Regulatory molecules can alter
activity of allosteric enzymes
INHIBITOR
ENZIM
Enzyme inhibition
For enzymes that obey Michaelis-Menten
laws, compounds that reversibly inhibit
enzyme activity can be kinetically
classified
Consider two general types:
Competitive
inhibitors
Noncompetitive inhibitors
Competitive vs.
noncompetitive
inhibitors
Competitive inhibitors: are
chemicals that resemble an
enzyme’s normal substrate and
compete with it for the active site.
site
Substrate
Competitive inhibitor
Enzyme
Noncompetitive inhibitors:
Inhibitors that do not enter the
active site,
site but bind to another
part of the enzyme causing the
enzyme to change its shape,
shape which
in turn alters the active site.
site
Substrate
active site
altered
Enzyme
Noncompetitive
Inhibitor
Competitive inhibitors
Y intercept the same regardless of whether
inhibitor is present or absent, BUT the slope
differs between the two lines
Competitive inhibitors
Do not alter Vmax
Increase Km
Competitive inhibition can be overcome by
increasing substrate concentration
Block substrate binding to the active site
of an enzyme
Examples of competitive inhibitors
Alcohol (alcohol dehydrogenase)
UpCA (RNase)
DHFR inhibitors (DNA metabolic inhibitor of
tumors)
Sulfa drugs (anti-bacterial drugs)
Physiological examples: feedback
inhibition, pancreatic trypsin inhibitor
Enzyme inhibition & automobile
antifreeze
Ethylene glycol (EG) is a constituent of
antifreeze
EG not toxic but is converted to oxalic acid
which form crystals in the kidneys leading
to extensive tissue damage and renal
failure
First step of conversion of EG to oxalic
acid is its oxidation to an aldehyde by
alcohol dehydrogenase
This reaction inhibited by ethanol which
competes with EG for binding to the
alcohol dehydrogenase
Inhibition of RNase
by UpCA
An example of a
typical competitive
inhibitor:
UpCA has a very
similar structure
to the genuine
substrate, but is
chemically unable
to undergo reaction.
Use of Enzyme inhibitors as anti-cancer drugs:
Folate (folic acid)
Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:
eventually leads to synthesis of thymine nucleotides (DNA metabolism)
Competitive inhibitors of dihydrofolate reductase used in cancer treatment
(resemble folate, bind ~1000x tighter):
Sulfa Drugs
Resemble PABA in
structure
Blocks metabolic
activity of bacteria
Examples of the Physiological (regulatory) Role of Enzyme Inhibitors
Feedback inhibition: The end-product of a biochemical pathway is similar to the
starting product and may (competitively) bind to and inhibit one of the enzymes
in the pathway:
Another example of regulatory competitive
inhibition: Inhibition
by Pancreatic Trypsin Inhibitor
Noncompetitive inhibitors
Plots converge on the X axis in the
presence or absence of inhibitor
Noncompetitive inhibitors
Do not alter Km
Decrease Vmax
Noncompetitive inhibition cannot be
overcome by adding excess substrate
Bind to a site outside of catalytic site of
enzyme and act by decreasing the
turnover number of an enzyme
In noncompetitive inhibition
why is Vmax decreased while
Km remains unchanged?
The inhibitor lowers the concentration of
functional enzyme
The remaining “uninhibited” enzyme behaves
like a more dilute solution of that enzyme
(assumes [inhibitor] is limiting)
In other words, the substrate can still bind to
enzyme alone or enzyme complexed with the
inhibitor. But only free enzyme will catalyze
the reaction.
Since the pool of free enzyme is lower in
presence of inhibitor, Vmax will also be lower
Irreversible Enzyme Inhibitors
Inhibitor becomes covalently linked to the
enzyme
Attachment often occurs at the active site
Examples: 5-fluorouracil, DIPF (nerve
gas), penicillin
Suicide Inhibitors
Irreversible enzyme inhibitors
Participate in the enzymatic reaction like the
substrate
At some point in the reaction they get ‘stuck’
and become permanently linked to the enzyme.
Example: 5-Fluorouracil, a suicide inhibitor
which targets thymidylate synthase and is used
in cancer treatement.
TS cannot catalyze
reaction
5Fluorouracil
Enzyme inhibitors as anti-bacterial drugs
Penicillin
Most Drugs
and
toxins are
enzyme
inhibitors:
Enzyme Inhibition – 1: Irreversible
Denaturation or
specific sites
Heavy metals
Usually try to avoid
EDTA in
buffers
Occasionally useful
experimentally
Hg-inactivation
If bind at active site:
Substrate
can
reduce rate of
irreversible inhibition
Rate(inhibition) vs.
[S] gives substrate
dissociation
constant, KS.
can
implicate Cys
Reversible Inhibition more interesting
Reversible inhibition
Competitive, non-competitive, mixed,
uncompetitive
Each suggestive of a different mechanism
Actually, operational definitions
Depend
on kinetic behavior
Point to be emphasized later
1. Competitive inhibition - 1
Poss. Mechanism: Binds in active site,
competing with substrate.
EI
Ki
S+E+
I
ES
E+P
Inhibition constant: in this
mechanism, this is a true
dissociation constant, because
EI is a “dead-end” complex
KM (apparent
dissociation, as ES can
progress…)
Competitive inhibition 2 - example
Succinate dehydrogenase
CO2-
CO2-
CH2
CH
CH2
CH
CO2-
CO2-
Succinate
CO2CH2
CO2malonate
Fumarate
Competitive inhibition 3: kinetics
Kinetics in the presence of many types of inhibitor
looks like Michaelis-Menton w/ modified constants:
app
Vmax
S
v app
;
K
M S
With competitive inhibition...
Vmax S
app
v
; Vmax
Vmax
K M 1 I K i S
K Mapp
Show the effect on a
double-reciprocal plot
Competitive inhibition 4 - plots
Kinetics show Vmax
unchanged
if [S] >> [I], drown out
inhibition
Affect (increase) KM
amount
of S required
for 1/2 Vmax
in
d hi
bi
te
Non-mathematically
1/v0
-1/KM
Slope =
KM/Vmax
1/Vmax
1/[S]
Competitive inhibition 5: mechanism
Previously suggested mechanism was one of
several that lead to similar kinetic effects
Perhaps
I is really an alternative substrate
Competitive inhibition defined operationally
Often
don’t know the mechanism
Any inhibitor that affects KM, but not Vmax
The “specificity” constant = kcat/KM is changed
Some
use “specific inhibition” to avoid mechanistic
implication
Operational definition
Lower Vmax, without changing KM.
affect rate w/o affect substrate-binding
Very small addition to active site
-1/KM
Vmax = kcat[E0] can be lowered by
reducing kcat - non-competitive inhibition
reducing amount of active enzyme - irreversible inhibition
Rare: difficult to affect kcat w/o affecting KM.
1/Vmax
e.g. H+ or cation.
1/[S]
Remote inhibitor binding that affects exact positioning of catalytic
groups
Difficult to distinguish from irreversible inhibition
1/v0
Possible mechanisms
in
d h ib i
te
2. Non-competitive inhibition
More commonly, component of “mixed inhibition”.
3. Mixed inhibition
Operational definition
Lower Vmax,
also raise KM, EIS more likely
to dissociate than ES
bi
te
d
1/v0
-1/KM
in
hi
1/Vmax
1/[S]
4. Uncompetitive inhibition
Operational: Km and Vmax are
changed by the same factor.
Possible mechanism: Inhibitor
te
i
binds to ES complex, not at all to 1/v hib
0 in
free-E.
d
1/Vmax
Naturally rare:
-1/K
Inhibition
of myo-inositol
monophosphatase by Li+ antidepressant
Experimental product inhibition
often uncompetitive
M
1/[S]
Classification of Inhibitors according to
effect on kinetic parameters
Vapp/ Vapp
KMapp
Competitive
Yes
No
i.e. only KM changed
Uncompetitive
No
Yes
Vmax
& KM changed by
corresponding amounts giving
/ / double- reciprocal plots
Mixed
Yes
Yes
Enzymes
Without Enzyme
With Enzyme
Free
Energy
Free energy of activation
Reactants
Products
Progress of the reaction
Active site involves amino acids far
apart in the primary sequence of a
protein (example: lysozyme)
Catalytic sites form clefts or
crevices
Substrate molecules bound within cleft
Water (unless involved in catalysis) is
normally excluded
Overall nonpolar character of cleft can
enhance binding of substrate
Cleft may also contain polar residues which
may take on catalytic properties within this
nonpolar microenvironment (exception to the
rule regarding hydrophobic “core” present in
many globular proteins)
Oleh:
Maria Ulfah, S.Si, M.Pd
JURUSAN PEND.BIOLOGI FPMIPA
IKIP PGRI SEMARANG
What Are Enzymes?
Most enzymes are
Proteins (tertiary
and quaternary
structures)
Act as Catalyst to
accelerates a reaction
Not permanently
changed in the
process
Enzymes
Are specific for
what they will
catalyze
Are Reusable
End in –ase
-Sucrase
-Lactase
-Maltase
Enzyme-Substrate Complex
The substance
(reactant) an
enzyme acts on is
the substrate
Substrate
Joins
Enzyme
Formation of an enzyme-substrate complex
How do enzymes
Work?
Enzymes work
by weakening
bonds which
lowers
activation
energy
Active Site
A restricted region of an enzyme
molecule which binds to the substrate
Active
Site
Substrate
Enzyme
The enzyme active site (features)
The catalytic site is relatively small compared
with the rest of the enzyme. Why are many
enzymes so big then?
The catalytic site is a three-dimensional entity
Substrates are bound to enzymes by multiple
weak, non-covalent interactions (electrostatic
bonds, hydrogen bonds, van der Waals
forces, hydrophobic interactions)
The specificity of binding depends on the
precisely defined arrangement of atoms in
an active site
Emil Fischer (over
100 years ago): came
up with the “lock and
key” hypothesis to
describe enzymesubstrate interactions
Active site of cytochrome P-450
Induced Fit
A change in
the shape of
an enzyme’s
active site
Induced by the
substrate
“Induced fit” model: a
more refined model
that takes into
account the enzyme
assumes a
complimentary shape
to that of its substrate
only after substrate
binds to the enzyme.
More dynamic
scenario compared to
the lock and key
hypothesis
Induced Fit
A change in the configuration of an
enzyme’s active site (H+ and ionic
bonds are involved)
Induced by the substrate
substrate
Active Site
Enzyme
induced fit
What Affects Enzyme Activity?
Three factors:
1. Environmental Conditions
2. Cofactors and Coenzymes
3. Enzyme Inhibitors
1. Environmental Conditions
1. Extreme Temperature are the most
dangerous
- high temps may denature (unfold) the
enzyme.
2. pH (most like 6 - 8 pH near neutral)
3. Ionic concentration (salt ions)
Environmental factors affecting enzyme activity
2. Cofactors and Coenzymes
Inorganic substances (zinc, iron)
and vitamins (respectively) are
sometimes need for proper
enzymatic activity.
activity
Example:
Iron must be present in the
quaternary structure hemoglobin in order for it to
pick up oxygen.
KINETIKA ENZIM
Michaelis-Menten model of
enzyme kinetics (Vmax & Km)
Key element in their model is the
existence of the ES complex
Rate of catalysis (V) increases with
increasing [S], where V is defined as the
number of moles of product formed per
second
When enzyme concentrations are constant, V is
linearly proportional to [S] WHEN [S] IS SMALL.
At high [S] (when S is in vast excess of the
[enzyme]), V is nearly independent of [S]
The Michaelis-Menten equation
Km & Vmax
Km = the Michaelis constant
Defined as the [substrate] at which the
reaction rate is half of its maximal value
Used to define relative affinity of an
enzyme for its substrate
The higher the Km value, the lower the
affinity
Vmax: describes the maximal rate of
product formation when [S] is high
Under such conditions all of the existing
“pool” of enzyme active sites are full
From Vmax an enzyme’s turnover
number can be determined (expressed
as the number of substrate molecules
converted into product per unit time)
Double-reciprocal
(lineweaver-Burk) plot
Used to calculate Km &
Vmax
Also used to characterize
mechanisms of enzyme
inhibition by specific
compounds
Data expressed as 1/V
versus 1/[S]: gives a
straight line
Calculating Km and Vmax
Allosteric enzymes do not conform
to Michaelis-Menten kinetics
Yield a sigmoidal curve on a V versus S
plot (not hyperbolic as seen under
Michaelis-Menten conditions)
Sigmoidal curve indicates cooperative
binding (binding of one molecule of S
affects affinity and binding of
additional S molecules)
Regulatory molecules can alter
activity of allosteric enzymes
INHIBITOR
ENZIM
Enzyme inhibition
For enzymes that obey Michaelis-Menten
laws, compounds that reversibly inhibit
enzyme activity can be kinetically
classified
Consider two general types:
Competitive
inhibitors
Noncompetitive inhibitors
Competitive vs.
noncompetitive
inhibitors
Competitive inhibitors: are
chemicals that resemble an
enzyme’s normal substrate and
compete with it for the active site.
site
Substrate
Competitive inhibitor
Enzyme
Noncompetitive inhibitors:
Inhibitors that do not enter the
active site,
site but bind to another
part of the enzyme causing the
enzyme to change its shape,
shape which
in turn alters the active site.
site
Substrate
active site
altered
Enzyme
Noncompetitive
Inhibitor
Competitive inhibitors
Y intercept the same regardless of whether
inhibitor is present or absent, BUT the slope
differs between the two lines
Competitive inhibitors
Do not alter Vmax
Increase Km
Competitive inhibition can be overcome by
increasing substrate concentration
Block substrate binding to the active site
of an enzyme
Examples of competitive inhibitors
Alcohol (alcohol dehydrogenase)
UpCA (RNase)
DHFR inhibitors (DNA metabolic inhibitor of
tumors)
Sulfa drugs (anti-bacterial drugs)
Physiological examples: feedback
inhibition, pancreatic trypsin inhibitor
Enzyme inhibition & automobile
antifreeze
Ethylene glycol (EG) is a constituent of
antifreeze
EG not toxic but is converted to oxalic acid
which form crystals in the kidneys leading
to extensive tissue damage and renal
failure
First step of conversion of EG to oxalic
acid is its oxidation to an aldehyde by
alcohol dehydrogenase
This reaction inhibited by ethanol which
competes with EG for binding to the
alcohol dehydrogenase
Inhibition of RNase
by UpCA
An example of a
typical competitive
inhibitor:
UpCA has a very
similar structure
to the genuine
substrate, but is
chemically unable
to undergo reaction.
Use of Enzyme inhibitors as anti-cancer drugs:
Folate (folic acid)
Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:
eventually leads to synthesis of thymine nucleotides (DNA metabolism)
Competitive inhibitors of dihydrofolate reductase used in cancer treatment
(resemble folate, bind ~1000x tighter):
Sulfa Drugs
Resemble PABA in
structure
Blocks metabolic
activity of bacteria
Examples of the Physiological (regulatory) Role of Enzyme Inhibitors
Feedback inhibition: The end-product of a biochemical pathway is similar to the
starting product and may (competitively) bind to and inhibit one of the enzymes
in the pathway:
Another example of regulatory competitive
inhibition: Inhibition
by Pancreatic Trypsin Inhibitor
Noncompetitive inhibitors
Plots converge on the X axis in the
presence or absence of inhibitor
Noncompetitive inhibitors
Do not alter Km
Decrease Vmax
Noncompetitive inhibition cannot be
overcome by adding excess substrate
Bind to a site outside of catalytic site of
enzyme and act by decreasing the
turnover number of an enzyme
In noncompetitive inhibition
why is Vmax decreased while
Km remains unchanged?
The inhibitor lowers the concentration of
functional enzyme
The remaining “uninhibited” enzyme behaves
like a more dilute solution of that enzyme
(assumes [inhibitor] is limiting)
In other words, the substrate can still bind to
enzyme alone or enzyme complexed with the
inhibitor. But only free enzyme will catalyze
the reaction.
Since the pool of free enzyme is lower in
presence of inhibitor, Vmax will also be lower
Irreversible Enzyme Inhibitors
Inhibitor becomes covalently linked to the
enzyme
Attachment often occurs at the active site
Examples: 5-fluorouracil, DIPF (nerve
gas), penicillin
Suicide Inhibitors
Irreversible enzyme inhibitors
Participate in the enzymatic reaction like the
substrate
At some point in the reaction they get ‘stuck’
and become permanently linked to the enzyme.
Example: 5-Fluorouracil, a suicide inhibitor
which targets thymidylate synthase and is used
in cancer treatement.
TS cannot catalyze
reaction
5Fluorouracil
Enzyme inhibitors as anti-bacterial drugs
Penicillin
Most Drugs
and
toxins are
enzyme
inhibitors:
Enzyme Inhibition – 1: Irreversible
Denaturation or
specific sites
Heavy metals
Usually try to avoid
EDTA in
buffers
Occasionally useful
experimentally
Hg-inactivation
If bind at active site:
Substrate
can
reduce rate of
irreversible inhibition
Rate(inhibition) vs.
[S] gives substrate
dissociation
constant, KS.
can
implicate Cys
Reversible Inhibition more interesting
Reversible inhibition
Competitive, non-competitive, mixed,
uncompetitive
Each suggestive of a different mechanism
Actually, operational definitions
Depend
on kinetic behavior
Point to be emphasized later
1. Competitive inhibition - 1
Poss. Mechanism: Binds in active site,
competing with substrate.
EI
Ki
S+E+
I
ES
E+P
Inhibition constant: in this
mechanism, this is a true
dissociation constant, because
EI is a “dead-end” complex
KM (apparent
dissociation, as ES can
progress…)
Competitive inhibition 2 - example
Succinate dehydrogenase
CO2-
CO2-
CH2
CH
CH2
CH
CO2-
CO2-
Succinate
CO2CH2
CO2malonate
Fumarate
Competitive inhibition 3: kinetics
Kinetics in the presence of many types of inhibitor
looks like Michaelis-Menton w/ modified constants:
app
Vmax
S
v app
;
K
M S
With competitive inhibition...
Vmax S
app
v
; Vmax
Vmax
K M 1 I K i S
K Mapp
Show the effect on a
double-reciprocal plot
Competitive inhibition 4 - plots
Kinetics show Vmax
unchanged
if [S] >> [I], drown out
inhibition
Affect (increase) KM
amount
of S required
for 1/2 Vmax
in
d hi
bi
te
Non-mathematically
1/v0
-1/KM
Slope =
KM/Vmax
1/Vmax
1/[S]
Competitive inhibition 5: mechanism
Previously suggested mechanism was one of
several that lead to similar kinetic effects
Perhaps
I is really an alternative substrate
Competitive inhibition defined operationally
Often
don’t know the mechanism
Any inhibitor that affects KM, but not Vmax
The “specificity” constant = kcat/KM is changed
Some
use “specific inhibition” to avoid mechanistic
implication
Operational definition
Lower Vmax, without changing KM.
affect rate w/o affect substrate-binding
Very small addition to active site
-1/KM
Vmax = kcat[E0] can be lowered by
reducing kcat - non-competitive inhibition
reducing amount of active enzyme - irreversible inhibition
Rare: difficult to affect kcat w/o affecting KM.
1/Vmax
e.g. H+ or cation.
1/[S]
Remote inhibitor binding that affects exact positioning of catalytic
groups
Difficult to distinguish from irreversible inhibition
1/v0
Possible mechanisms
in
d h ib i
te
2. Non-competitive inhibition
More commonly, component of “mixed inhibition”.
3. Mixed inhibition
Operational definition
Lower Vmax,
also raise KM, EIS more likely
to dissociate than ES
bi
te
d
1/v0
-1/KM
in
hi
1/Vmax
1/[S]
4. Uncompetitive inhibition
Operational: Km and Vmax are
changed by the same factor.
Possible mechanism: Inhibitor
te
i
binds to ES complex, not at all to 1/v hib
0 in
free-E.
d
1/Vmax
Naturally rare:
-1/K
Inhibition
of myo-inositol
monophosphatase by Li+ antidepressant
Experimental product inhibition
often uncompetitive
M
1/[S]
Classification of Inhibitors according to
effect on kinetic parameters
Vapp/ Vapp
KMapp
Competitive
Yes
No
i.e. only KM changed
Uncompetitive
No
Yes
Vmax
& KM changed by
corresponding amounts giving
/ / double- reciprocal plots
Mixed
Yes
Yes
Enzymes
Without Enzyme
With Enzyme
Free
Energy
Free energy of activation
Reactants
Products
Progress of the reaction
Active site involves amino acids far
apart in the primary sequence of a
protein (example: lysozyme)
Catalytic sites form clefts or
crevices
Substrate molecules bound within cleft
Water (unless involved in catalysis) is
normally excluded
Overall nonpolar character of cleft can
enhance binding of substrate
Cleft may also contain polar residues which
may take on catalytic properties within this
nonpolar microenvironment (exception to the
rule regarding hydrophobic “core” present in
many globular proteins)