Biochemistry 3511

Enzyme Catalysis:Chapter 11, Sections 1-3

1. General properties of enzymes
Enzymes are classified by the type of reactions
Enzymes act on specific substrates
Some enzymes require cofactors
2. Activation energy and reaction coordinate
3. Catalytic mechanisms
Acid-base catalysis
Covalent catalysis
Metal ion catalysis
Proximity-assisted catalysis
Transition state theory

General Properties of Enzymes

1. Higher reaction rates:
up to 106-1012 times
greater.
2. Milder reaction conditions:
pH around
neutral, atmospheric pressure, temperature <
100oC.
3. Greater reaction specificity:
rarely have
side products.
4. Capacity for regulation:
allosteric control,
covalent
modification, variation in expression.

General Properties of Enzymes

mild reaction
conditions:
Nitrogenase
N2 + 8H+ + 8e- + 16ATP
2NH3 + H2 + 16ADP + 16Pi
Nitrogenase

Iron
catalyst

Pressure

1 atm

200 atm

Temperature

room
temperature

300-550oC
PDB-ID: 1N2C

Enzyme Reaction Types

most are specific,

but a few are
not

Specificity (Mostly) Due to Selective Binding

geometric and electronic

specificity

stereospecificity

Specificity (Mostly) Due to Selective Binding

(citrate is prochiral)

stereospecificity

Two General Models for Binding Specificity

1. Lock and Key
2. Induced Fit

PDB-ID: 2HU4

Active site fits substrate

like a glove.
Charge, H-bonding,
hydrophobicity and shape
features can often be
calculated and binding
constants predicted.

Two General Models for Binding Specificity

1. Lock and Key
2. Induced Fit

Substrate binding induces

specific conformational shift.
Difficult to calculate/predict.
More common than
believed.

Some Enzymes Require Cofactors

Cofactors: enzymes chemical teeth
(holo = present,
apo = absent)

(organic molecules)
(Cu2+, Fe3+, Zn2+)

(transiently associated)

(permanently associated)

Cosubstrate Examples: NAD+/NADP+

Some Enzymes Require Cofactors

Cofactors: enzymes chemical teeth
(holo = present,
apo = absent)

(organic molecules)
(Cu2+, Fe3+, Zn2+)

(transiently associated)

(permanently associated)

Prosthetic Group Example:

Heme

Transition State
(Reaction Coordinate) Diagrams

HA-HB + HC HA + HB-HC

= free energy of activation

Greaction = Gproducts - Greactants Greaction

Greaction = 0

A+BP+ Q

<0

Transition State
(Reaction Coordinate) Diagrams

AI P

blue curve: A rate-limiting

red curve: I rate-limiting
Which path is more favorable?
(more negative G)
Neither! Greaction same.

Effect of a Catalyst on
Transition State Diagrams

Overall
Greaction
not affected!

Free energy of
activation (G)
lower.

Acid-Base Catalysis
1. Acid-base catalysis
Amino acid side-chains donate or accept protons and
participate in chemical reactions.

RNA Hydrolysis (Chemical)

Acid-Base Catalysis
RNA Hydrolysis by Rnase A (Enzymatic)

Enzymes often have a concerted mechanism.

What other amino acids are candidates for acid-base catalysis?

Acid-Base Catalysis
Amino Acids Involved in Acid-Base Catalysis

Acid-Base Catalysis
RNA Hydrolysis by Rnase A (Enzymatic)

The acid and base must be regenerated at the conclusion of

the catalytic reaction. There cannot be overall changes to
the enzyme.

Acid-Base Catalysis and pH Dependence

The pK values of the two His residues
were measured to be 5.4 and 6.4.
Which pK value belongs to each His?
(Assume we are at pH = 7.)
His 12 (5.4), and His 119 (6.4)
fumarase of TCA
cycle

What would happen at pH = 5?

What about pH = 9?

Acid-Base Catalysis
Lowers the Energy of the Transition State

Mitigates charge
development in the
transition state,
thereby stabilizing it.

Covalent Catalysis
2. Covalent catalysis
Nucleophiles catalyze reactions through the transient formation
of covalent bonds with substrates.

In the case of this serine protease, the covalent bond stabilizes

the transition state.

Red = substrate

The substrate is covalently

bound to the enzyme via serine.

What other amino acids can be used covalent catalysis?

Covalent Catalysis
Nucleophiles/Electrophiles
Involved in Covalent Catalysis

Covalent Catalysis:
Acetoacetate Decarboxylation

Metal Ion Catalysis

3. Metal ion catalysis
The unique electronic properties of the metal ion facilitate the reaction

Metal ions participate in the catalytic process in three major ways:

1. By binding to substrates to orient them properly for reaction.
2. By mediating oxidation-reduction reactions through reversible
changes in the metal ions oxidation state.
3. By electrostatically stabilizing or shielding negative charges.
In many metal-ion catalyzed reactions, the metal ion acts in much
the same way as a proton to neutralize negative charge.
However, metal ions can be present at neutral pH at higher
concentrations, and have charges > +1.

Metal Ion Catalysis:

Carbonic
Anhydrase

intermediate-bound

Proximity-Assisted Catalysis
Non-enzymatic
4. Proximity-assisted catalysis
Enzymes accelerate reactions by bringing reacting
groups together and orienting them for reaction.

Intramolecular reaction 24x faster!

Enzymatic

Proximity-Assisted Catalysis

Proximity accounts for up to 5-fold rate enhancement.

Correct geometric orientation accounts for up to 100-fold
rate enhancement.

Proximity-Assisted Catalysis
Enzymatic
Correct geometric orientation accounts for up to 100-fold
rate enhancement.

Enzymatic

Proximity-Assisted Catalysis

Proximity accounts for up to 5-fold rate enhancement.

Correct geometric orientation accounts for up to 100-fold
rate enhancement.
Freezing out rotational and translational motion
accounts for up to 107-fold rate
enhancement!!!

Enzymatic

Proximity-Assisted Catalysis

Proximity accounts for up to 5-fold rate enhancement.

Correct geometric orientation accounts for up to 100-fold
rate enhancement.
Freezing out rotational and translational motion
accounts for up to 107-fold rate
enhancement!!!
Electrostatic stabilization of the transition state in the
active site and removal of water from the active site also
important.

Proximity-Assisted Catalysis:
Entropically Favorable
Consider uncatalyzed bimolecular reactions:
2 free reactants single restricted transition state
conversion is entropically unfavorable.
Consider uncatalyzed unimolecular reactions:
flexible reactant rigid (more conformationally restricted) transition state
conversion is also entropically unfavorable.
Enzyme-catalyzed reactions:
enzyme uses binding energy of substrates to organize reactants into the
more restricted enzyme-substrate (ES) complex.

entropy cost is paid during binding

rigid reactant complex transition state conversion is
entropically OK

5. Transition state theory

Transition state stabilization can
significantly lower the activation
energy for a reaction.

Transition State
Theory: Enzymes Bind
Transition States

Enzyme active sites are complimentary to the transition state of the reaction being catalyzed

Transition State Theory:

Enzymes Bind Transition States
Enzyme active sites are complimentary to the transition state of the
reaction being catalyzed.
Enzymes bind transition states better than substrates or products.
Stronger interaction with than ground state lowers the activation
barrier.
This is largely an enthalpic effect.

Transition State Analogs

Are Enzyme Inhibitors
proline racemase

transition state
analogs