Enzyme Specificity
Lecture 3
Objective
To understand Specificity of enzymes
Specificity means:
• Ability of an enzyme to catalyse a specific
reaction and NO others
The active site
• The active site, is a
special region where
catalysis occurs
• Occupies small part of
enzyme volume
• The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed more
easily
Closer Look at Active site
•AS is not a point or line on enzyme
•It is a region in enzyme molecule where catalysis occurs
•AS has binding site and a catalytic site
•AS has a 3D Structure; residues widely separated in the
primary structure are brought closer in the AS
•Clefts or crevices
AS: General Characteristics
• Substrates bound by multiple weak interactions
• AS include both polar and nonpolar amino acids and
create hydrophilic and hydrophobic
microenvironment
• Specificity depends on precise arrangement of
atoms in active site
• Substrates bound by multiple weak interactions
• Specificity depends on precise arrangement of
atoms in active site
Enzyme
Amino acid in active site
Hexokinase
His
Phosphoglucomutase
Ser
Trypsin
Ser, His
Carbonic anhydrase
Cysteine
Carboxypeptidase
His, Arg, Tyr
Thrombin
Ser, His
Aldolase
Lys
Chymotrypsin
Ser, his
Choline esterase
Ser
Cofactors
• An additional non-protein
molecule that is needed by
some enzymes to help the
reaction
• Tightly bound cofactors are
called prosthetic groups
• Cofactors that are bound
and released easily are
called coenzymes
• Many vitamins are
coenzymes
Nitrogenase enzyme with Fe, Mo and ADP cofactors
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
The Substrate
• The substrate of an enzyme are the reactants
that are activated by the enzyme
• Enzymes are specific to their substrates
• The specificity is determined by the active site
• Enzyme specificity can be for the type of reaction
it catalyses or for its choice of substrates
• Substrate has a bond or linkage that can be
attacked by the enzyme
Active site
At the active site, functional groups of the
enzyme interact with substrate (eg: RNase A)
This interaction may weaken one of its bonds or
participate in a reaction by transforming an
electron or proton
(eg: catalytic mechanism of Serine proteases)
• Enzyme and substrate binding releases
‘binding energy’.
• This free energy is used by enzyme to lower
the activation energy of reaction and it
provides specificity to reaction.
Chemical Reaction Pathway
Making reactions go faster
• Biological systems are very sensitive to temperature
changes
• Increasing the temperature make molecules move
faster
• Enzymes can increase the rate of reactions without
increasing the temperature
• They do this by lowering the activation energy
• They create a new reaction pathway “a short cut”
An enzyme controlled pathway
Most Enzyme controlled reactions proceed 108 to 1011 times
faster than corresponding non-enzymic reactions
Substrate binding site consists of an indentation or cleft
on the surface of an enzyme. This cleft is complementary
in shape to the substrate (geometric complementarity)
The amino acid residues that form the binding site are
arranged to interact specifically with the substrate in an
attractive manner (electronic complementarity)
Two Models
• Lock and Key
• Induced Fit
The Lock and Key Hypothesis
• Proposed by Emil Fisher in 1894
• the active site exists “pre-formed” in the enzyme prior to
interaction with the substrate
• Fit between the substrate and the active site of the enzyme is
exact , like a key fits into a lock very precisely
• The key is analogous to the enzyme and the substrate
analogous to the lock
• Temporary structure called the enzyme-substrate complex
formed
• Products have a different shape from the substrate
• Once formed, they are released from the active site, leaving it
free to become attached to another substrate
The Lock and Key Hypothesis
S
E
E
E
Enzymesubstrate
complex
Enzyme may
be used again
P
P
Reaction coordinate
The Lock and Key Hypothesis
• This explains enzyme specificity
• This explains the loss of activity when
enzymes denature
The Induced Fit Hypothesis
• Some proteins can change their shape (conformation)
• When a substrate combines with an enzyme, it induces a
change in the enzyme’s conformation
• The active site is then moulded into a precise
conformation
• Making the chemical environment suitable for the
reaction
• The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
This model was proposed by
Daniel Koshland in 1958
It requires the active site to be
floppy and substrate to be rigid
Chemical Specificity
1. Group Specificity
• Enzymes may act on several different, though
closely related substrates
• They catalyse reaction involving a particular
group (eg: ALDH)
• ALDH catalyses the oxidation of variety of
alcohols
• HK: assist the transfer of PO4 from ATP to
several different hexose sugars
2. Absolute specificity
• Enzyme acting only on one particular substrate
• Eg: Glucokinase catalyses the transfer of PO4
from ATP to glucose and to no other sugars
(other egs: urease, arginase, catalase)
3. Steriochemical specificity/
Geometric specificity
• Substrate chemically identical
with different arrangement of
atoms in in 3D space
• Only one of the isomers undergo
reaction by a particular enzyme
L-amino acid
L-Amino acid Oxidase
Ketoacid
Trypsin acts only on polypeptide containing Lamino acids, not those containing of D-amino
acids
Enzymes of glucose metabolism are specific for
D-glucose
Yeast Alcohol Dehydrogenase (YADH) oxidises
ethanol to aldehydes
YADH acts on Methanol at 25 fold slower
YADH acts on Propanol 2.5 fold slower
NADPH (differ in a PO4 at 2’ from NADH) does
not bind to YADH
• Glycerol Kinase phosphorylates glycerol to
Glycerol-3-P
• If phosphorylation occurs at C1, the product is Dglycerol -3-P
• If phosphorylation occurs at C3, the product is Lglycerol -3-P
• The enzyme always produces only L-isomer
• Identical chemical groups in a substrate become
different after binding at the microenvironment
of the active site of the enzyme (1948, A.G. Ogston)
Enzyme Binding Sites
• Active Site = Binding Site + Catalytic Site
• Regulatory Site: a second binding site, occupation
of which by an effector or regulatory molecule,
can affect the active site and thus alter the
efficiency of catalysis – improve or inhibit
Identification and
Characterization of Active Site
• Structure: size, shape, charges, etc.
• Composition: identify amino acids
involved in binding and catalysis.
Binding or Positioning Site
(Trypsin)
H2O
O
N
NH
arginine
or
lysine
CH
C
NH
C
"long + side chain"
+
complementary binding
or positioning site
_
"SPECIFICITY"
Binding or Positioning Site
(Chymotrypsin)
H2O
O
N
NH
phenylalanine
tyrosine
tryptophan
CH
C
O
NH
C
"aromatic side chain"
complementary binding
or positioning site
Hydrophobic
Pocket
"SPECIFICITY"
Catalytic Site
(e.g. Chymotrypsin)
H2 O
O
N
NH CH
C
NH
C
O
catalytic site
complementary
"CATALYSIS"
Probing the Structure of the Active
Site
Model Substrates
Model Substrates
(Chymotrypsin)
H2 O(ROH)
peptide bond
O
N
NH CH
aromatic
side chain
R
C
NH
C
acyl transfer to H2 O
Peptide Chain?
O
H3N
CH
NH
C
C
R
or
O
H3N
NH
CH
C
NH2 (or -OCH3)
R
or
O
H3N
CH
C
NH2 (or -OCH3)
R
All Good Substrates!
a-amino group?
O
H 2C
R
C
NH2
(OCH 3)
Good Substrate!
Side Chain Substitutions
Good Substrates
CH3
CH3
CH3
Cyclohexyl
t-butyl-
Conclusion
Bulky Hydrophobic Binding Site
O
Y
CH
C
X
X,Y = various
= hydrophobic
positioning
group
"Hydrophobic Acyl Group Transferase"
Probing the Structure of the Active
Site
Competitive Inhibitors
Arginase
H2N
+
NH2
C
H2O
+
NH
(CH2)3
+
H3N
CH
NH2
H2N
NH3
+
C
(CH2)3
COO
arginine
+
H3N
CH
COO
ornithine
O
urea
Good Competitive Inhibitors
+
NH3
NH2
CH
+
+
H3N
NH3
NH3
(
(CH
2)3
(
(CH
2)4
CH
NH
+
COO
ornithine
+
H3N
CH
lysine
O
COO
(
(CH
2)2
+
H3N
CH
COO
canavanine
-
Poor Competitive Inhibitors
+
+
H3 N
+
NH3
NH3
CH3
(CH 2)3
(CH2)3
(CH2 )3
CH 2
putrescine
(l,4-diaminobutane)
H 2C
COO
-
4-aminovaleric acid
All Three Charged Groups are Important
+
H3N
CH
COO
-
a-aminovaleric acid
Conclusion
Active Site Structure of Arginase
-
binding
site
+
-
catalytic
site
Identifying Active Site Amino Acid
Residues
Covalent Inactivation
F
CH3
CH O
CH3
P
CH3
O
CH
O
CH3
Diisopropyl Phosphofluoridate
Inactivates Chymotrypsin by forming a 1:1 covalent
adduct to Serine195.
Iodoacetic acid inactivates Ribonuclease by reacting
with His12 and His119.
Affinity Labeling
(General Approach)
Binding Site
X
X
+
Positioning Group
Reactive Group
Y
Affinity Labeling
(Tosyl-L-phenylalanine chloromethylketone)
CH3
O
O
S
O
NH O
CH2 CH C CH2 Cl
Positioning
Group
Reactive
Group
Inactivates Chymotrypsin by forming a 1:1
covalent adduct to Histidine57
Trapping of Enzyme-Bound Intermediate
(Chymotrypsin)
O
CT
CH2
O
OH + O2N
Ser195
O
C
CH3
p-nitrophenylacetate
O2N
O
O–
p-nitrophenol
O
CT
CH2
O
C
CH3
"acyl" enzyme
stable at pH 3
Implicates Ser195 in catalytic mechanism.
Mechanism