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Fundamentals of
Biochemistry
Third Edition
Donald Voet • Judith G. Voet •
Charlotte W. Pratt
Chapter 11
Enzymatic Catalysis
Copyright © 2008 by John Wiley & Sons, Inc.
Properties of Enzyme Catalysts
• massive reaction rate enhancements
• increase by factors of 106 – 1014 typical
• specificity
• stereochemical selectivity for substrates
and products
• including chirality (i.e. enantiomeric
selectivity)
• regulation by various compounds is
common
• generally not as robust to severe
environmental conditions compared to
nonbiological catalysts
Examples of Enzyme Rate Enhancements
Table 11-1
Enzyme classification (E.C. system)
1. Oxidoreductases
Oxidation–reduction reactions
First number (1-6)
indicates reaction class
2. Transferases
Transfer of functional groups
3. Hydrolases
Hydrolysis reactions
4. Lyases
Group elimination to form double bonds
5. Isomerases
Isomerization
6. Ligases
Bond formation coupled with ATP hydrolysis
Subclasses of the Oxidoreductase class (1)
1. 1. -.1. 2. -.1. 3. -.1. 4. -.1. 5. -.1. 6. -.1. 7. -.1. 8. -.1. 9. -.1.10. -.1.11. -.1.12. -.1.13. -.1.14. -.1.15. -.1.16. -.1.17. -.1.18. -.1.19. -.1.20. -.1.21. -.1.97. -.-
Acting on the CH-OH group of donors.
Acting on the aldehyde or oxo group of donors.
Second number
Acting on the CH-CH group of donors.
gives subclass,
Acting on the CH-NH(2) group of donors.
provides more
Acting on the CH-NH group of donors.
specificity (e.g.
Acting on NADH or NADPH.
details about the
Acting on other nitrogenous compounds as donors.
Acting on a sulfur group of donors.
type of
Acting on a heme group of donors.
substrate/product)
Acting on diphenols and related substances as donors.
Acting on a peroxide as acceptor.
Acting on hydrogen as donor.
Acting on single donors with incorporation of molecular oxygen.
Acting on paired donors, with incorporation or reduction of molecular oxygen.
Acting on superoxide as acceptor.
Oxidizing metal ions.
Acting on CH or CH(2) groups.
Acting on iron-sulfur proteins as donors.
Acting on reduced flavodoxin as donor.
Acting on phosphorus or arsenic in donors.
Acting on x-H and y-H to form an x-y bond.
Other oxidoreductases.
Subclasses and sub-subclasses of the Isomerase
class (5)
5. -. -.- Isomerases.
5. 1. -.- Racemases and epimerases.
5. 1. 1.- Acting on amino acids and derivatives.
5. 1. 2.- Acting on hydroxy acids and derivatives.
5. 1. 3.- Acting on carbohydrates and derivatives.
5. 1.99.- Acting on other compounds.
5. 2. -.- Cis-trans-isomerases.
5. 3. -.- Intramolecular oxidoreductases.
5. 3. 1.- Interconverting aldoses and ketoses, and related compounds.
5. 3. 2.- Interconverting keto- and enol- groups.
5. 3. 3.- Transposing C==C bonds.
5. 3. 4.- Transposing S-S bonds.
5. 3.99.- Other intramolecular oxidoreductases.
5. 4. -.- Intramolecular transferases (mutases).
5. 4. 1.- Transferring acyl groups.
5. 4. 2.- Phosphotransferases (phosphomutases).
5. 4. 3.- Transferring amino groups.
5. 4. 4.- Transferring hydroxy groups.
5. 4.99.- Transferring other groups.
5. 5. -.- Intramolecular lyases.
5.99. -.- Other isomerases.
Enzyme specificities
• mainly non-covalent forces (H-bonds,
electrostatics, van der Waals, hydrophobic)
• typically involves a binding cleft
• complimentary in shape to substrate(s) {see caveat later}
• resulting desolvation of the substrate is sometimes an
important feature
• binding cleft is typically pre-formed to a large
degree
• ‘lock and key’ idea: Emil Fischer, 1894
• on the contrary, sometimes the cleft needs to
change conformation substantially to fit the
substrate:
• ‘induced fit’ idea: Dan Koshland
• energetic disadvantage (some binding energy spent to ‘cause’
binding cleft to reconfigure)
• advantage: binding site will not do catalysis accidentally,
reactive groups not situated correctly until substrate binds
A cartoon illustrating binding specificity in
an active site cleft
multiple sites of
interaction are usually
involved
Figure 11-1
Stereo and enantiomeric selectivity in
enzyme reactions
• able to distinguish between natural and
unnatural enantiomers of a molecule
• able to introduce a new chiral center with
enantiomeric specificity
• important for ensuring the production and
recognition of the correct biological
molecules
• a major advantage over most non-biological
catalysts employed organic chemistry
application
• example: the enzyme aconitase
Aconitase introduces a two new chiral
centers when the hydroxyl shifts
*
*
* new chiral centers
Page 325
illustrates how multiple attachment
points to a chiral surface make it
possible to discriminate between the
two chemically identical (-CH2-COO-)
substituents on the central carbon; the
hydroxyl gets shifted specifically
towards one of them and not the other
Enzymes are usually not perfectly
selective
• some enzymes are intentionally nonspecific
• e.g. proteolytical degradation in the gut
• imperfect selectivity of most enzymes
makes it possible to design a substratemimic that can be detected
spectrophotometrically
• very useful in assaying enzyme activities
• requires some change in absorption (magnitude
or wavelength) between substrate and product
Page 325
The range of possible enzyme reactions is
expanded greatly by the use of bound cofactors
organic
transiently bound,
cycling on-off
Page 325
bound persistently
(e.g. covalent)
Involvement of cofactors in enzyme
reactions
• direct involvement in the catalytic events
• cofactor sometimes left unaltered at the
end of the reaction
• or, sometimes the cofactor is altered (e.g.
oxidized/reduced)
• cofactor sometimes recycled ‘in-place’, by the
activity of another enzyme acting on the bound
cofactor
• cofactor sometimes cycled off the enzyme; it is
replaced by a ‘fresh’ cofactor, and then
regenerated by another enzyme in solution
Page 325
NAD+/NADH: an example of a ubiquitous redox
cofactor. Often acting as a ‘co-substrate’ (i.e.
recycled off the enzyme)
note the
introduction of a
chiral center
Page 325
Definition
• apo-enzyme: enzyme with no cofactor
bound
• holo-enzyme: enzyme with cofactor bound
Page 325
Transition state theory for reaction rates
(Henry Eyring)
Example with a very
simple reaction:
Along a reaction
pathway, one passes
(very briefly) through a
maximally unstable
configuration: this is
the ‘transition state’
In a simple reaction, the reaction coordinate can be understood in
terms of a specific geometric feature of the reaction (e.g. here, the
Ha-Hb distance). In more complex reactions, the reaction
coordinate is sometimes less tangible.
A transition state diagram for a reaction
involving a change in free energy between
substrates and products
Height of the barrier that
must be overcome is
called the free enegy of
activation. The free
energy of activation (in
the forward direction) is
shown.
The rate of a reaction is governed by how well-populated the
transition state (shown here as X#) is.
Dependence of reaction rate on free energy
of activation
The rate of a reaction
is governed by how
well-populated the
transition state
(shown here as X#) is.
Specifically, the rate depends on e(-DG#/RT)
Lower DG# means faster rate.
By thermodynamic
arguments, the
degree to which X# is
populated depends on
how much higher its
energy is than the
substrates. (i.e. it
depends on DG#)
N.B. Intermediates are not the same as transition
states. In a multi-step reaction mechanism, there will
be intermediates, and transition states between every
stable (or marginally stable) species.
Figure 11-6
Catalysis of a reaction by lowering the
transition state energy
The rate enhancement
goes as e-(DDG#/RT)
Note that forward and
reverse rates are
increased by the same
factor; the equilibrium
constant and K for the
reaction are unchanged
Figure 11-7
factor of 1,000,000
corresponds to about 34Kcal/mol (smaller than
the energy of a covalent
bond, comparable to a
few strong non-covalent
interactions
Acid-Base catalysis: one of the dominant
features in enzyme mechanisms
acid catalysis
base catalysis
a ‘concerted’ acid-base mechanism would involve both features
Figure 11-8
Acid-Base catalysis: one of the dominant
features in enzyme mechanisms
• participation of multiple side chains (or
cofactors makes concerted acid-base
mechanisms possible (and common)
for enzymes
• the use of acid-base mechanisms is
one reason the activities of most
enzymes are pH dependence
• details of the pH dependence can
often be used to infer something
about what functional groups are
involved in the mechanism
Figure 11-8
Ribonuclease (RNase):
an example of acidbase catalysis
cleavage of a nucleic acid
between the phosphate and
the 3’ sugar hydroxyl
pH dependence suggests
participation of groups with
pK’s of 5.4 and 6.4
(understood to be His12
and His119 based on crystal
structure)
Figure 11-10
Ribonuclease (RNase): an example of acid-base
catalysis
Step 1: the 2’OH belonging to the substrate acts as the nucleophile.
His12 acts as general base to activate the nucleophile. His119 acts as a
general acid, donating a proton to the leaving group 5’OH.
Figure 11-10 part 1
Ribonuclease (RNase): an example of acid-base
catalysis
Step 2: H2O comes in to act as the nucleophile in the second step,
attacking the same phosphate as before. Now, His119 acts as a general
base and His12 acts as a general acid, protonating the leaving group
(the 2’OH). The two steps are a simple reversal, including the roles of
the side chains. Only the substrate participants are different (H20 vs
sugar 5’OH).
Figure 11-10 part 1
Covalent catalysis
• typically involves formation of a covalently bonded
intermediate between the substrate and either a
protein sde chain or cofactor
• ex: acetoacetate decarboxylation by a primary amine
Figure 11-8
Common nucleophiles (and electrophiles)
Figure 11-12
Metal ions in catalysis
• common in redox reactions
• substrate binding
• stabilization of negative charges
stabilization of hydroxy ion (not ordinarily present
at high concentrations) for nucleophilic attack
example: carbonic anhydrase (Zn2+)
Figure 11-13b
Metal ions in catalysis
Zn provides a hydroxyl ligand
for attacking CO2
Figure 11-13b