Enzymes

Chapter 8

Important Group of Proteins

• Catalytic power can incr rates of rxn > 106

• Specific

• Often regulated to control catalysis

• Coupling biological pathway

Catalysis Happens…• Enzymes use many intermolecular forces

– At enzyme active site

– From R grps of aa’s

• Substrates brought together

• Optimal orientation

• Making/breaking bonds facilitated

– Transition state stabilization

– Allows high energy transition state

• Enzyme native conformation crucial

Additional Chemical Components

• Prosthetic Groups

– Cofactors (Table 8-1)

– Coenzymes (Table 8-2)

• Bound to apoenzyme

holoenzyme

Table 8-1

Table 8-2

Rxns Occur at Enzyme Active Sites (8-1)

• Physical clefts

• “Lined” w/ aa funct’l grps

• Stabilize transition state S P

• Complex ES forms (reversible)

Fig. 8-1

Energetics• For any (cat’d) rxn involving S: G = H - T S

G:

– If negative

– If = 0

– If positive

G:

– Depends on free energy prod’s – free energy reactants

– Independent on path of rxn (so catalysis doesn’t alter)

– No info on rate of rxn

For S < == > P at Equilibrium

• Keq = [P] / [S]

G = G’o + RT ln [P] / [S], and

G = 0, so

G’o = - RT ln [P] / [S]

G’o = - RT ln Keq’

– So Keq directly related to G for rxn (Table 8-4)

Table 8-4

G’o = diff in free energy between S, P

• Enzymes do NOT effect Keq’, G’o

• Enzymes impt when energy must be added for rxn to proceed

S* = Transition State = High Energy Intermediate• Must add energy for S <==> S*

• Common rxn intermediate

• “Fleeting molecular moment”

• Can go to S or P (8-2)

G*(SP) = Activation Energy

– Diff in energy S to S*

– Enzymes lower G*

Fig. 8-2

ES* = Enzyme Substrate Complex

• Must add energy for E + S < == > ES*

• BUT less energy

• So lower rxn pathway

• Can go to E + S or E + P (8-3)

• Note: E is always regenerated

G*(cat’d)

– Diff in energy S to ES*

– So rxn more energetically favorable in presence of catalyst

Fig. 8-3

Enzymes Effect Rxn Rate

• Use rate constant (k) to describe rate

S < == > P

• Velocity (V) of rxn dependent on [S], k

– V = k [S]

– First order rxn

• Can relate k to G*

– Eq’n 8-6

– Relationship between k and G* is inverse and exponential

Table 8-5

Summary

• Enzymes don’t change overall energy difference, equilibrium

• Enzymes do lower EA

• Enzymes do increase k

Source of Energy from within Enzyme to Facilitate Rxn S <==> P• Most impt: ES complex

• Existence proven experimentally, theoretically

• Enzyme active site

– Aa residues directly participate (catalytic grps)

– Only small part of total volume

– Catalytic grps may be far apart in primary structure

• Folding is important!

Fig. 8-4

Substrate Binding to Enzyme Active Site

• Multiple weak interactions

– What are these?

• Must have proper orientation between atoms

• Substrate, active site have complementary shapes

• Commonly crevice is nonpolar

– Polar residues at site commonly participate

– Water excluded unless it participates

• So: microenvironment w/ aa funct’l grps that have particular properties essential for catalysis of rxn

Binding Specificity

• DNA evolution protein w/ optimal aa sequence optimal E/S interactions lowering energy nec for rxn

• So, depends on precisely arranged atoms in active site

Two theories of E/S “match” • Lock & key (Fisher, 1894) (8-4)

– If precise match to S, why S* or P?

• Complementarity to S*

– Enz active site complementary to transition state

– So weak interactions encourage S*, then stabilize it

• Best energetically when S* fits best into enz active site

– Must expend energy for rxn to take place

– BUT overall many weak interactions lower net activation energy

• E/S “match” also confers specificity

Fig. 8-5

Fig. 8-5 - cont’d

Other Factors that Reduce Activation Energy

• Besides multiple weak atom-atom interactions

• Physical, thermodynamic factors influence energy, rate of catalyzed rxn

– Entropy reduction (8-7)

• S held in proper orientation

• Don’t rely on random, productive collisions

Fig. 8-7

– Desolvation

• H-bonds between S and solvent decreased

• Incr’s productive collisions

– Induced fit

• Enzyme conformation changes when S binds

• Brings impt funct’l grps to proper sites

• Now has enhanced catalytic abilities