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

but a few are not .Enzyme Reaction Types most are specific.

Specificity (Mostly) Due to Selective Binding geometric and electronic specificity stereospecificity .

Specificity (Mostly) Due to Selective Binding (citrate is prochiral) stereospecificity .

•Charge. Induced Fit PDB-ID: 2HU4 •Active site fits substrate “like a glove”.Two General Models for Binding Specificity 1. H-bonding. . “Lock and Key” 2. hydrophobicity and shape features can often be calculated and binding constants predicted.

•Difficult to calculate/predict. “Lock and Key” 2.Two General Models for Binding Specificity 1. . •More common than believed. Induced Fit •Substrate binding induces specific conformational shift.

apo = absent) (organic molecules) (Cu2+. Fe3+.Some Enzymes Require Cofactors Cofactors: enzymes’ “chemical teeth” (holo = present. Zn2+) (transiently associated) (permanently associated) .

Cosubstrate Examples: NAD+/NADP+ .

Fe3+.Some Enzymes Require Cofactors Cofactors: enzymes’ “chemical teeth” (holo = present. Zn2+) (transiently associated) (permanently associated) . apo = absent) (organic molecules) (Cu2+.

Prosthetic Group Example: Heme .

Greactants Greaction ‡   Greaction = 0 A+BP+ Q <0 .Transition State (Reaction Coordinate) Diagrams   HA-HB + HC  HA + HB-HC   = free energy of activation Greaction = Gproducts .

Transition State (Reaction Coordinate) Diagrams   AI  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. .

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

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 .

. There cannot be overall changes to the enzyme.Acid-Base Catalysis RNA Hydrolysis by Rnase A (Enzymatic) The acid and base must be regenerated at the conclusion of the catalytic reaction.

4).) His 12 (5.Acid-Base Catalysis and pH Dependence The pK values of the two His residues were measured to be 5. Which pK value belongs to each His? (Assume we are at pH = 7. and His 119 (6.4) fumarase of TCA cycle What would happen at pH = 5? What about pH = 9? .4.4 and 6.

.Acid-Base Catalysis Lowers the Energy of the Transition State Mitigates charge development in the transition state. thereby stabilizing it.

the covalent bond stabilizes the transition state. Red = substrate The substrate is covalently bound to the enzyme via serine. Covalent catalysis Nucleophiles catalyze reactions through the transient formation of covalent bonds with substrates. What other amino acids can be used covalent catalysis? . In the case of this serine protease.Covalent Catalysis 2.

Covalent Catalysis Nucleophiles/Electrophiles Involved in Covalent Catalysis .

Covalent Catalysis: Acetoacetate Decarboxylation .

. 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. In many metal-ion catalyzed reactions. metal ions can be present at neutral pH at higher concentrations. By mediating oxidation-reduction reactions through reversible changes in the metal ion’s oxidation state.Metal Ion Catalysis 3. However. 3. the metal ion acts in much the same way as a proton to neutralize negative charge. By electrostatically stabilizing or shielding negative charges. 2. 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! .

• Correct geometric orientation accounts for up to 100-fold rate enhancement. .Enzymatic Proximity-Assisted Catalysis • Proximity accounts for up to 5-fold rate enhancement.

.Proximity-Assisted Catalysis Enzymatic • Correct geometric orientation accounts for up to 100-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.

• 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. • Correct geometric orientation accounts for up to 100-fold rate enhancement. .Enzymatic Proximity-Assisted Catalysis • Proximity accounts for up to 5-fold rate enhancement.

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

Transition state theory Transition state stabilization can significantly lower the activation energy for a reaction.5. Transition State Theory: Enzymes Bind Transition States Enzyme active sites are complimentary to the transition state of the reaction being catalyzed .

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

Transition State Analogs Are Enzyme Inhibitors proline racemase transition state analogs .