UCSB CHEM 109C
Dr. Kalju Kahn
Catalysis
Learning goals:
To have some understanding about:
¾ The fundamentals of chemical catalysis
¾ Different ways that enzymes carry out catalysis
¾ The concept of transition state stabilization
Images: active sites of chymotrypsin and alcohol dehydrogenase
What are Enzymes?
• Enzymes are catalytically active biological macromolecules
• Most enzymes are globular proteins,
however some RNA (ribozymes, and ribosomal RNA) and
even polysaccharides have catalytic properties
• Study of enzymatic processes is the oldest field of biochemistry, dating
back to late 1700s
• Study of enzymes has dominated biochemistry in the past and
continues to do so
Why Biocatalysis?
• Higher reaction rates
• Greater reaction specificity
• Milder reaction conditions
• Capacity for regulation
Image: Bad Boys of the Arctic - Polar Bears by Thomas D. Mangelsen
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“Life” without Enzymes: Slow
Source: Wolfenden, Acc. Chem. Res. 2001, 34, 938-945
“Life” without Enzymes: Mess
COO
-
COO
NH2
O
OH
COO
O
COO
COO
-
-
COO
-
OH
COO
-
-
COO
O
OH
NH2
Metabolites have many potential pathways of decomposition
More: http://www-chem.ucdavis.edu/groups/toney/Chorismate.html
Enzymatic Reaction Specificity
COO
-
COO
O
-
OH
COO
-
-
COO
O
OH
Chorismate mutase
Image: http://www.cstl.nist.gov/div831/biotech/techact/binding.html
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Enzymatic Stereospecificity
Stereospecific abstraction of hydride by alcohol dehydrogenase
H
HO
D
O
Pro-R
position
H
Pro-R
hydrogen is
removed by ADH
Enzymatic Substrate Selectivity
OH
H
-
H
+
OOC
NH3
OOC
H
-
No binding
+
NH3
OOC
+
NH3
OH
HO
OH
H
H
H
NH
Binding but no reaction
CH3
Example: Phenylalanine hydroxylase
Enzyme-Substrate Complex:
• Enzymes act by binding substrates
– the non-covalent enzyme substrate complex is
known as the Michaelis complex
– allows thinking in terms of chemical interactions
– allows development of kinetic equations
v=
kcat [ E ]total [ S ]
K m + [S ]
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Lock-and-Key Model
• Proposed by Emil Fisher in 1894 to explain
the high specificity of enzymes
• Enzyme’s binding pocket is pre-shaped to
accept only the substrate
Induced Fit Model
• Conformational changes may occur upon ligand binding
(Daniel Koshland in 1958).
– This adaptation is called the induced fit.
– Induced fit allows tighter binding of the ligand
– Induced fit can increase the affinity of the protein for a
second ligand
• Both the ligand and the protein can change their
conformations
+
Induced Fit in Hexokinase
4
Rate Acceleration
• The enzyme lowers the activation barrier compared
to the aqueous reaction
• Enzyme converts one
difficult step to many
easy steps
• In theory, the enzyme may
also facilitate the tunneling
through the barrier. This may
be important for electrons.
Barrier lowering
Barrier thinning
How to Lower ΔG≠?
Enzymes bind
transition states best
• The idea was proposed by Linus Pauling in 1946:
– enzyme active sites are complimentary to the transition
state of the reaction
– enzymes bind transition states better than substrates
– stronger interactions with the transition state as
compared to the ground state lower the activation barrier
More about Linus Pauling: http://www.paulingexhibit.org/
How is TS Stabilization Achieved?
– acid-base catalysis: give and take protons
– covalent catalysis: change reaction paths
– metal ion catalysis: use redox cofactors, pKa shifters
– electrostatic catalysis: preferential interactions with TS
5
Acid-base Catalysis: Chemical Example
Consider ester hydrolysis:
O
O
+
R
H-OH
O
R
O CH3
OH
O
+ H
+
+
R
CH3OH
OH
CH3
Water is a poor nucleophile, and methanol is a poor leaving group
Aqueous hydrolysis can be catalyzed either by acids or by bases
Enzymes can do acid and base catalysis simultaneously
General Acid-Base Catalysis
• Example: ketosteroid isomerase
• Converts one steroid to another
– androst-5-ene-3,17-dione to androst-4-ene-3,17-dione
– product is “Andro” (the first commercial testosterone prohormone)
• Abstracts proton from the sp3 carbon
– difficult reaction (C–H proton has high pKa)
– reaction proceeds via enolate intermediate
– similar to the reaction of triosephosphate isomerase
Enolate intermediate
Ketosteroid Isomerase
Mechanism
O
Tyr 14
OH
O
H
H
O
Step 1:
H
• Proton abstraction by Asp 38
• Hydrogen bond to stabilize the
enolate intermediate
O
Asp 38
O
Tyr 14
OH
-
O
H
H
O
H
O
Tyr 14
O
OH
Asp 38
Step 2:
• Proton donation by Asp 38
O
H H
H
O
O
Asp 38
6
Covalent Catalysis: Chemical Example
O
O
O
H2O
CH3
O
H3C
O
O
H3C
slow
+
-
-
+
2 H
+
O
O
O
CH3
O
H3C
O
+
N
fast
CH3
+
H3C
..
..
H
O
H
O
O
..
+
H3C
O
+
N
-
-
• Hydrolysis is accelerated
because of charge loss in
the transition state
makes pyridine a good
leaving group.
CH3
OH
H
O
• The anhydride hydrolysis
reaction is catalyzed by
pyridine, a better
nucleophile than water
(pKa=5.5).
N
N
O
H3C
+
Covalent Catalysis: In Enzymes
• Proteases and peptidases
– chymotrypsin, elastase, subtilisin
– reactive serine nucleophile
• Some aldehyde dehydrogenase
– glyceraldehyde-3phosphate dehydrogenase
– reactive thiolate nucleophile
• Aldolases and decarboxylases
– amine nucleophile
• Dehalogenases
– carboxylate nucleophile
NH
2
-
HO
O
S
-
O
N
O
N
O
N
O
N
O
General Acid-Base + Covalent Catalysis:
Cleavage of Peptidoglycan by Lysozyme
From the X-ray structure, it is known that the C-1 carbon is located between two
carboxylate residues of the protein (Glu-35 and Asp-52). Asp-52 exists in its ionized
form, while Glu-35 is protonated.
Asp-52 acts as a nucleophile
to attack the anomeric
carbon.
Glu-35 acts as a general acid
and protonates the leaving
group in the transition state.
Water hydrolyzes the
covalent glycosyl-enzyme
intermediate
Glu-35 acts as a general
base to deprotonate water in
the transition state of the
hydrolysis step.
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