Bi515 Chapter 6, part 1

Enzymes: Overview
2010
Assigned problems: CH6 (4th edition)
# 3,4,6,7,8,9,10,11,16,18
5th edition: Ch 6, # 3,4,6,8,11-13, 21, 23
 Important Terms

• Catalyst (usually > 10^6 times faster)--affects

forward and reverse rates, no effect on Keq
• Specificity, Substrate, Active Site
• Cofactor (inorganic) vs. Coenzyme
(organic/metalloorganic)
• Prosthetic group (covalently or tightly bound)
• Holoenzyme vs. apoenzyme/apoprotein (minus its
coenzyme/cofactor)
Coenzymes
Enzyme Classification
 Enzymes affect Reaction Rates, not
Equilibria (6-2)

Std. Free
energy
change, pH 7

S  [TS]  P
• Free energy of [P] – [S] = negative: favorable
reaction. This determines Keq.
• This doesn’t mean rxn will be fast!
• Size of energy barrier (activation energy)
determines rate
• Activation energy: barrier to aligning substrates
and enzyme, forming transient bonds, breaking
others, reaching transition state
Free energy of substrates and products
determines the Keq

Keq = [P]/[S]
•If Keq = 1000, are there more products or substrates at
equilibrium?
•For this reaction, which molecule has the lowest free energy:
S or P?
•What is the Keq for the reverse reaction?
 Transition State

• Not a stable intermediate like ES, EP

• Point at which decomposition to substrate or to
product is equally favored
• Difference in energy between ground state and
transition state (activation energy) determines
reaction rate, i.e. the smaller the energy difference
the faster the reaction.
• Catalysts lower activation energy so enhance rate
of forward and reverse reactions.
 Enzyme catalyzed vs Uncatalyzed
Reactions (6-3)

E + S  ES  EP  E + P
 Rate-Limiting Step (R.D.S.)

• Step that determines overall rate of enzyme

• Step with highest activation energy
• Which one?
 Why not lower activation energies for all
reactions?

•If activation energy is low, easy for

molecule to react.
•Would result in loss of complex
macromolecules and gain of simpler forms.
Higher the barrier, the more stable the
molecule.
•Enzymes selectively lower activation energy
barriers, for their specific rxn, not all rxns
 In a cell…
• Reactions rarely reach equilibrium
• Products are used up in metabolic pathway
or substrates are limiting
• Rate determined by [S] and by k:
– V=k[S] (first order reaction) or V=k[S1][S2]
(second order reaction for S1+S2 P)
– k = rate constant; k is inversely proportional to
activation energy (exponential relationship)
How do enzymes get energy to catalyze reactions?

1) They bind in multiple locations to S and TS;

lowers activation energy, and increases binding
energy

2) Most interactions occur in transition state

-stabilize distribution of charges in t.s.

3) If TS is more stable (lower in free energy),

then energy to reach TS (activation energy) is
lowered.
ES highly stabilized (low free energy) due to multiple
interactions with S. So stable that activation energy is
extremely high, reaction is impeded.
ES is stabilized, but even
more binding sites to t.s.
(binding energy, Gb).
Transition state is stabilized
Energy to bend stick is
partially compensated with
gain of binding energy to t.s.
 Specificity derives literally from binding
energy: groups on enzyme are arranged to
bind to transition state. Substrate that is
different will have a lower binding energy, so
the activation energy is not lowered as much.

How would you

draw the reaction
profile for a poor
substrate?
 Opposing Forces to think about
for enzyme-substrate interactions

• Why is activation energy so high?

– How does binding energy overcome these obstacles?
• Loss of entropy in aligning substrates
– Substrates held in proper orientation, so collide. Takes rare occurrence and increases the
chances of collision/reaction greatly. See 6-7
• Need to disrupt solvation shell of water molecules that are
H-bonded to substrate
– Formation of substrate-enzyme bonds releases enzyme-water bonds
• Substrates must be distorted to reach t.s.
– Binding energy helps compensate for energy required for distortion
• Enzyme must be properly aligned to react
– Induced fit brings functional groups on enzyme close enough to react with substrate, also
adds binding interactions
Rate enhancement by entropy reduction
 Try this
• Urease enhances the rate of urea hydrolysis by
1014. If a given amount of urease hydrolyzes a
given amount of urea in 5 min, how long would it
take to hydrolyze that amount of urea without
urease?
• When hexokinase is heated at 45C, it loses 50%
of its activity after 12 min. If hexokinase is
incubated with its substrate and then heated, it
loses only 3% activity in 12 min. Suggest why the
thermal denaturation of hexokinase was retarded in
the presence of one of its substrates.