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•The energy requiring step from substrate to transition state is an energy
barrier called the free energy of activation ∆G‡
•Transition state is the unstable (10-13 seconds) highest energy species on
the reaction coordinate
•Enzymes lower the energy of activation barrier by lowering the energy of
the transition state (stabilization) to allow for transformation to occur
•The active site of enzymes are thought to be structurally similar and bind
tighter to the transition state compared with the ground state substrate
Today:
Acid-base catalysis
Electrophilic catalysis
Covalent catalysis
Principles of Catalysis
•Uncatalyzed reactions, even when thermodynamically favorable
(spontaneous), often are extremely slow.
•They are slow because of the height of the activation energy
needed to reach the transition state
•Activation energy is high because the formation of the transition
state is unfavorable due to the presence of unstable + and - charges
that develop.
•Stabilization of these charges will lower the activation energy and
accelerate the rate of the reaction (active site pit)
Principles of Enzyme Catalysis
Uncatalyzed reactions are too slow due to energetic transition states
(very large ∆G‡)
• Analogous reactions found in organic chemistry are
observed in Enzymology
Acid Base Catalysis - Donation or abstraction of protons
Covalent Catalysis - Covalent enzyme-substrate
intermediate
Metal Ion Catalysis- Substrates and metals positioned for
reaction
Electrostatic Considerations- Compliment of charges
with transition state
Proximity and Orientation - Substrates aligned for
reaction
Types of Enzyme Catalysis
Acid Base Catalysis - Donation or abstraction of protons
Covalent Catalysis - Covalent enzyme-substrate
intermediate
Metal Ion Catalysis (Electrostatic)- Substrates and
metals positioned for reaction
Acid-Base Catalysis
• General acids transfer protons
• General bases abstract protons
• Specific acid or base catalysis is when the proton
or hydroxide ion is the catalyst (organic)
Acid-Base Catalysis
R
C
R
O
C- O
CH2
CH2
H
H+
Ketone
Transition
state
R
C
OH
CH2
Enol
Uncatalyzed reaction occurs very slowly due to unfavorable carbanion
Acid-Base Catalysis
R
C
O
HA
R
R
O- H+ A-
C
CH2
CH2
H
H+
..
C
OH + HA
CH2
B
Ketone
Transition
state
Enol
Acid-Base Catalysis
•Amino Acid side chains that can act as acid-base
catalysts:
Asp, Glu, His, Lys, Cys, Tyr
•Acid - base reactions are governed by sidechain pKa’s
•Catalysis often sensitive to pH changes (pKa-e.g.)
• pH - rate profiles can distinguish between acid-base
catalysis and lead to the identification of participating
catalytic residues (mutagenesis)
Electrostatic Catalysis
When a charged transition state cannot be stabilized by an acid-base
catalyst (e.g. no ionization) the charge can be neutralized by an
oppositely charged group from the catalyst (active site of enzyme)
Amino Acid side chains that participate in electrostatic catalysis:
Asp, Glu, His, Lys, Arg
In an enzyme’s active site several electrostatic interactions (also
known as ion-pairs or salt bridges) can collectively attract the
substrate into the active site pocket (sometimes followed by
Ground state destabilization) and stabilize the transition state
contributing to reduction in activation barrier.
Orotidine 5’-monophosphate decarboxylase
And Ground State Destabilization
Wu, Ning et al. (2000) Proc. Natl. Acad. Sci. USA 97, 2017-2022
Orotidine 5’-monophosphate decarboxylase
Orotidine 5’-monophosphate decarboxylase
And Ground State Destabilization
Wu, Ning et al. (2000) Proc. Natl. Acad. Sci. USA 97, 2017-2022
Metal Ion Catalysis
A specific type of electrostatic catalysis
Employs the positively charged metal ion to stabilize negative
charges for increased catalysis (also called Electrophilic catalysis)
Coordination of the cobalt complex increases the ability of a
nucleophile to catalyze the hydrolysis of glycine ester two
million fold
Metal Ion Catalysis
Another common role for metal catalysis is the interaction of
the metal ion with the side chain groups of the enzyme to
promote the reactivity of the enzyme’s groups through
electrostatic effects
Examples include metalloenzymes (urease)-important for
maintaining proper structure of protein and active site
residues
Also enzymes that accept co-factors in metal form (kinases)
MgATP (allows ATP to bind as neutral molecule)
Covalent Catalysis
A covalent bond is formed between the enzyme and its substrate
during the formation of the transition state
Covalent bond is initiated by an electron rich group in the active
site
Covalent catalysis involves a two part reaction process containing
two energy barriers in the reaction coordinate diagram
Transition
states
G
intermediate
reactant
product
Reaction coordinate
Somewhat stable
(isolate)
Nucleophilic groups in enzymes
Nucleophilic
group
Amino Acid
Intermediate
formed
OH
serine
acyl or phosphoryl
enzyme
SH
cysteine
acyl or phosphoryl
enzyme
COO-
aspartic acid
acyl enzyme
NH2
lysine
Schiff base
imidazole
histidine
acyl enzyme
OH
tyrosine
phosphoryl enzyme
In their deprotonated forms they attack electron deficient centres to form
covalent intermediates (pH dependent)
Many of the same groups that make good nucleophilic catalysts are good
acid-base catalysts because they contain unshared electron pairs
Acid-Nucleophilic Catalysis
Acid- -glucosidase
2nd messenger
Mutations in enzyme cause Gaucher disease, a lysosomal
storage disease (congested lysosomes) (defective spleens, liver,
neurological output)
Acid-Nucleophilic Catalysis
Acid-Nucleophilic Catalysis
Acid-Nucleophilic Catalysis
Metal ions as Covalent Catalysts- Carbonic anydrase
CO2
+
H2O
HCO3- +
H+
Metal ion covalent catalysis- Carbonic anydrase
CO2
+
H2O
HCO3- +
H+
Next
Nucleophilic catalysis
Microscopic reversibility and kinetic equivalence