BIOC 460, Spring 2008

Lecture 10
Enzymes: Introduction

Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 8, pp. 205-
217 (These pages in textbook are very important -- concepts of
thermodynamics are fundamental to all of biochemistry.)
Thermodynamics practice problems (same as for Lecture 2):
http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/ThermoPracticeProblems_08.pdf
(also linked in lecture notes directory)
Enzymes introduction sample problems:
http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC10_EnzIntrod/EnzIntrodSampleProblems.pdf

(also linked in lecture notes directory)

Key Concepts
• Enzymes are biological catalysts, very powerful and very specific.
– Enzymes increase rates of (bio)chemical reactions but have no
effect on Keq (and no effect on overall ΔG) of the reaction.
• Some enzymes need cofactors (inorganic ions or organic/metalloorganic
coenzymes, derived from vitamins) for their catalytic activities.
– Different cofactors are useful for different kinds of chemical reactions,
including transfers of specific kinds of groups or transfers of
electrons.
• Kinetics: the study of reaction rates. Rates depend on rate constants.
– Rate constants depend inversely and exponentially on Arrhenius
activation energy, ΔG‡, the difference in free energy between free
energy of transition state and free energy of reactant(s).
– Rate constants are increased by catalysts (enzymes), because
enzymes decrease ΔG‡.
– Enzymes lower ΔG‡ by affecting either ΔH‡ or ΔS‡ (or both).
– One way enzymes reduce ΔG‡ is by tight binding (noncovalent) of the
transition state.
– Enzymes generally change the pathways by which reactions occur.
– Rate enhancement (factor by which enzyme increases the rate of
a reaction) is determined by ΔΔG‡, the decrease in ΔG‡ brought
about by enzyme compared with uncatalyzed reaction's ΔG‡.

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Learning Objectives
(See also posted Thermo and Enzymes-Introduction sample problems.)
• Terminology: rate enhancement, cofactor, coenzyme, apoenzyme,
holoenzyme, prosthetic group, catalyst, activation energy, transition
state. (Review: equilibrium constant, mass action ratio for a reaction,
biochemical standard conditions, standard free energy change, actual
free energy change).
• Describe the general properties of enzymes as catalysts that are
especially important for their roles as biological catalysts.
• Explain the effect of a catalyst on the rate of a reaction, and on the
equilibrium constant of a reaction.
• Define "standard free energy change" and give the symbol for that
parameter.
• Write the mathematical expression relating ΔG°' to Keq', and be able to
interconvert ΔG°' and Keq'.
• Calculate the actual free energy change (ΔG'), given the starting
concentrations of appropriate chemical species and either ΔG°' or Keq'.
• Describe the relation between ΔG' and the rate of a reaction; using ΔG',
predict reaction direction.

Learning Objectives, continued

• Express the velocity of a simple reaction in terms of the rate constant
and the concentration of the reactant.
• Express the equilibrium constant of a reaction in terms of the equilibrium
mass action ratio.
• Express the equilibrium constant of a reaction in terms of the rate
constants for the forward and reverse directions. (Note that equilibrium
constants are symbolized with upper case K and rate constants with
lower case k.)
• If an enzyme increases the rate constant for the forward reaction by a
factor of 108, by what factor does it increase the rate constant for the
back reaction? What is the rate enhancement brought about by the
catalyst for that reaction?
• Draw the free energy diagram of a hypothetical reaction and show how a
catalyst may increase the rate of the reaction, pointing out on the
diagram ΔG for the overall reaction, ΔG‡uncat, and ΔG‡cat.
• Indicate (and name) the quantity on a free energy diagram (HINT: it's a
specific kind of ΔG) that determines the magnitude of the rate constant
for the reaction at a given temperature. You don't have to memorize the
equation relating this quantity to k.
• What reaction parameter (kinetic parameter) do enzymes affect in order
to increase the rate?

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Enzymes: Introduction
• Enzymes are proteins.
– (ribozymes: catalytic RNA molecules)
• biological catalysts
– not chemically altered in reaction
– do not change equilibrium constant (Keq) for reaction
– increase rate of reaction by providing a pathway of lower
activation energy to get from reactants to products
– operate under physiological conditions (moderate temps., around
neutral pH, low conc. in aqueous environment)
– work by forming complexes with their substrates (binding), thus
providing unique microenvironment for reaction to proceed, the
active site

– VERY HIGH SPECIFICITY for both reaction catalyzed and substrate

used
– VERY HIGH CATALYTIC EFFICIENCY
– ACTIVITIES of some enzymes REGULATED

CATALYTIC POWER OF ENZYMES

• RATE ENHANCEMENT = catalyzed rate constant/uncatalyzed rate
constant = factor by which catalyst increases rate of reaction
• Examples:

Berg et al., Table 8-1

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SPECIFICITY OF ENZYMES
• Enzymes very specific
– for substrate acted upon
– for reaction catalyzed
• Example: Proteases are a whole class of enzymes that all catalyze
hydrolysis of peptide bonds:

Substrate Specificity -- proteases as an example

• Substrate specificity (e.g., of proteases) due to precise interaction of
enzyme with substrate
– result of 3-D structure of enzyme active site where substrate has
to bind and be properly oriented for catalysis to occur
(A) Trypsin catalyzes hydrolysis
of peptide bonds on carboxyl
side of Lys and Arg residues
(digestive function in small
intestine, cleaves just about
any protein it encounters after
(eventually) every Lys and Arg)
(B) Thrombin (involved in blood
clotting cascade) catalyzes
hydrolysis of peptide bonds
between Arg and Gly residues
in specific sequences in
specific protein substrates
(activated only where blood
needs to clot, works only on
very specific target protein)
Berg et al., Fig. 8-1

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Enzyme Specificity, continued

• substrate specificity of proteases --
another example, chymotrypsin:
– cleaves on carboxyl side of aromatic and hydrophobic amino acid
residues
– evolutionarily related to trypsin
– Genes for trypsin and chymotrypsin are homologous.
• Ancestral gene duplicated and sequences diverged through
evolution.
• Substrate specificities for site of cleavage diverged, but catalytic
mechanism and overall tertiary structure was conserved.

Specificity of reaction catalyzed:

Many proteases also catalyze hydrolysis of carboxylic ester bonds:

Cofactors
• Some enzymes need cofactors for their activity.
• COFACTORS: small organic or metalloorganic molecules (coenzymes)
or metal ions
• Cofactors can bind tightly or weakly to enzymes. (Equilibrium below
can lie far to left, weak binding, or far to right, tight binding.)

• Prosthetic groups (e.g. heme in hemoglobin): tightly bound cofactors

(either coenzymes or metals)
– remain associated with their enzymes even between reaction cycles.
• Weakly bound coenzymes (which are NOT prosthetic groups) can
associate and dissociate from enzymes between reaction cycles, behaving
like substrates
– sometimes referred to as "cosubstrates"

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Common Coenzymes and Reactions They Mediate

See also Berg et al., Table 8.2 (for reference, not for memorization)

Coenzyme (precursor/vitamin) Reaction Mediated (Group Transferred)

Biotin Carboxylation (CO2)

Cobalamin (B12) Alkylation (methyl group), intramolecular

rearrangements, and ribonucleotide reduction

Coenzyme A (pantothenate) Acyl transfer (R–C=O group)

Flavin coenzymes (B2) Oxidation-reduction (hydrogen atoms) (1 or 2 e– transfer)

Lipoic acid Acyl group transfer

Nicotinamide coenzymes (niacin) Oxidation reduction (hydride ions H:–, 2 e– transfers)

Pyridoxal phosphate (B6) Amino group transfer (and many other reactions)

Tetrahydrofolate (folic acid) One-carbon transfer

Thiamine pyrophosphate (B1) Aldehyde transfer

Uridine diphosphate [UDP] Sugar transfer (hexose units)

Review Biological Thermodynamics

(Thermodynamics will not be covered again in class. Go back over notes for Lecture
2, and Berg, Tymoczko & Stryer, 6th ed. Chapter 1, pp. 11-12 and Chapter 8,
pp. 208-211)
• ΔG = ΔH – TΔS
• ΔG = Gproducts (final state) – Greactants (initial state)
• If ΔG < 0, reaction proceeds left to right (as written)
• reaction is exergonic, free energy decreases in going from reactants
to products)
• If ΔG = 0, reaction is at equilibrium (no net reaction occurs in either
direction)
• If ΔG > 0, reaction proceeds right to left (reverse direction from what's
written)
• Forward reaction would be endergonic, requiring INPUT of free
energy to go left to right.
• ΔGforward reaction = – ΔGreverse reaction
– Just change sign of ΔG for reversing direction.
• To get a reaction for which ΔG > 0 (a reaction with a positive ΔG) to go
forward, couple it with a reaction for which ΔG < 0 (negative ΔG) to
"drive" the process. (Exergonic reaction provides free energy "input" to
drive endergonic reaction.)

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Review thermodynamics (continued)

• Any free energy change, from any starting conditions, can be

described by the important equation:

is the actual mass action ratio, NOT Keq

(unless actual m.a. ratio happens to be the
equilibrium ratio, in which case ΔG’ = 0).

• ACTUAL free energy change ΔG' for any reaction or process

depends on 3 things:
1. standard free energy change for that reaction (ΔG°', a "reference”
telling where equilibrium lies)
2. actual starting concentrations of reactants and products
(mass action ratio)
3. temperature

CHEMICAL KINETICS (review from gen chem)

• For the reaction
• k = rate constant
• NOTE:
Rate constants are lower case k's.
Equilibrium constants are upper case K's.

• Velocity (rate) of forward reaction = vF = kF [S]eq

• Velocity (rate) of reverse reaction = vR = kR [P]eq
• At equilibrium, vF = vR, so kF [S]eq = kR [P]eq .
• The equilibrium constant is

• NOTE: Enzymes do NOT alter Keq.

• As catalysts, enzymes DO increase rate constants and thus increase rates
of reactions.
• COROLLARY: An enzyme that increases kF by a factor of 1010 must also
increase kR by a factor of 1010.
• There are a few Enzymes-Introd sample problems posted online.

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TRANSITION STATE THEORY

• transition state: an activated complex at the highest free
energy point on the reaction coordinate a PEAK on the free
energy diagram
• not isolatable as structures (lifetimes ~10–13 sec) -- they’re "in
transition", sort of with bonds half-made, half-broken.
• Chemical example: an SN2 reaction, attack of a thiolate anion on
iodoacetate: transition state (in brackets)(‡): a trigonal
bipyramid, with 3 covalent bonds + 2 more "half" bonds:

FREE ENERGY DIAGRAM FOR THE REACTION S → P

• free energy G vs. progress of reaction (i.e., the "reaction coordinate")
• Enzymes decrease
activation energy (ΔG‡)
for reactions they
catalyze.
• ΔG = overall difference in
free energy between
final (P) and starting (S),
not affected by enzyme.
• RATE of reaction IS affected
by enzyme. RATE depends
on ΔG‡, the Arrhenius
activation energy (i.e., the
free energy of activation
for the reaction).

Berg et al., Fig. 8.3

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Dependence of rate constant on ΔG‡, the activation energy

• Rate constant (k) depends on ΔG‡, the Arrhenius activation energy
(i.e., the free energy of activation for the reaction)
• ΔG‡ = G‡ – GS = difference in free energy between transition state
and starting state (S in this case), the "barrier" over which the reaction
must go in order to proceed.
• ΔG‡ has POSITIVE values (ΔG‡ > 0) -- it's a free energy BARRIER.
• k is rate constant for the reaction.
• κ is Boltzmann’s constant and
h is Planck’s constant.
• NOTE: Rate constant k is inversely
and exponentially dependent
on the activation energy, ΔG‡.
Velocity of the reaction:
(rate constant k is what’s inside large brackets.)

How could you increase the

reaction rate of S → P?

How could you increase the reaction rate of S → P?

• Rate of S → P = velocity = k [S]
to increase rate,
1) increase concentration of a reactant [S], or
2) increase the rate constant – HOW?
a) increase temperature, or
b) decrease ΔG‡ (catalyst)
• Enzymes increase reaction rates by decreasing ΔG‡ and thus increasing k.

How do enzymes
increase k, i.e.,
how do they
decrease ΔG‡?
step1 step2 step3
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed., Fig. 6-3

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How do enzymes increase k (decrease ΔG‡)?

• by changing the pathway of reaction, and
• by tightly binding transition state(s).
• “New pathways”: often multiple steps in an enzyme-catalyzed reaction.
•Intermediates are
troughs between
steps on free energy
diagram (e.g., ES and
EP).
•Each step has a
transition state (peak),
so each step has its
own ΔG‡.
step1 step2 step3
•Slowest step in pathway
(the "rate-limiting step")
= step with highest ΔG‡.

NOTE: Even the highest ΔG‡ (step #2 in figure above, ES < == > EP) for a
catalyzed reaction is less than the ΔG‡ for an uncatalyzed reaction.
Keq is not affected by the catalyst, and ΔG' is not affected by the catalyst.

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