Enzyme Catalysis I

Formation of the Transition State

Great White Shark (Carcharodon carcharias)

How Enzymes accelerate rates of reaction

we understand that enzymes lower activation energy required for reaction but there
are several components to enzyme behaviour and all of these increase reaction rates:
Substrate Binding
decreases random distribution of reactants (Proximity effect) thus increases energy
associated with system, thus less additional energy required to attain transition state
Stabilization of Transition State
reactants are orientated in a geometry that promotes transition state formation
transition state is stabilized, thus lowering activation energy
Facilitate Electron Flow
chemical reactions involve making and breaking of chemical bonds and these involve
transfer of electrons
amino acid side chains and co-factors (metal ions, co-enzymes, and water) participate
in electron flow pathway

Enzyme-Substrate Binding
enzyme catalysis begins with substrate binding to enzyme

E+S

fast

slow

k1

k2

k-1

ES

E+P

binding energy is equivalent to the free energy released

in the formation of a large number of weak interactions between
substrate and enzyme
binding energy serves two purposes:
establishes substrate specificity and proximity
increases catalytic efficiency
only the correct substrate can participate in most or all of the specific weak
interactions with the enzyme and thus maximize binding energy
catalytic efficiency is increased by lowering activation energy

The Proximity Effect

consider these non-enzyme catalyzed reactions

1.

2.

3.

4.

Effect of Substrate Binding and Stabilization of Transition State

on energy profile of reaction

note that activation energy is lowered in two ways

Enzymes stabilize transition states

all catalysts stabilize the transition state in a reaction
transition states are unstable arrangements of atoms in which chemical bonds
are in the process of being made or broken
enzymes stabilize the transition state by making the maximum number of
weak interactions with atoms in the transition state species
that is, the enzyme interacts better with the transition state form than it does
with either the original substrate(s) or the eventual product(s)
Stabilization of the transition state
lowers the activation energy required
for product formation

consider the reaction:

bromomethane + hydroxide anions methanol and bromine anions
bond being
formed

bond being
broken

progress to product formation requires that the transition state be formed.

many reactions proceed slowly in the absence of catalyst because formation of
the transition state arrangement is infrequent

Some reactions occur in multiple steps

some reactions occur in multiple steps involving multiple transitions states and
intermediates
transition states are high energy, extremely unstable and short-lived (10-13s).
Intermediates are more stable than transitions states, can often be detected
chemically, but still have a higher energy and are more unstable than either
the reactants or the products

Moran pg 165

Transition state (TS) analogues mimic true transition states

TS analogues are stable molecules that bind very strongly to enzymes because they
mimic the transition state (bind more strongly than the reactant/substrate)
though they are not perfect matches to the actual transition state, they give insight
into what the transition state must look like
TS analogs are useful for determining likely structural features of an enzyme when
bound to the true transition state.
TS analogs cannot be catalytically converted to product, therefore the enzyme
is trapped in the TS analog-bound state and is inhibited.

many inhibitors of enzymes are TS analogues and fall into the category of
competitive inhibitors (although not all competitive inhibitors are TS analogues)

Proteins consist of flexible elements and any structural representation is a snapshot

in time.

Phosphoglycerate kinase (PGK) catalyses the seventh step in glycolysis and is the first
enzyme in the pathway to produce energy, rather than consume it, by the creation of
adenosine triphosphate (ATP).

1,3-biphosphoglycerate + ADP

3-phosphoglycerate + ATP

ADP

1,3-biphosphoglycerate

open conformation
http://www.youtube.com/watch?v=dM9MMmHlZJs

closed conformation

Transition state analogs are often potent enzyme inhibitors and have various
therapeutic uses

Cholesterol lowering drugs

Statins (like Lipitor) are

cholesterol lowering drugs
because they are transitionstate analog inhibitors of
an enzyme called HMG-CoA
Reductase, a key enzyme in
the biosynthetic pathway for
cholesterol.

Antiviral Drugs
Tamiflu - Influenza Neuraminidase Inhibitor

H1N1
H1N5
etc

The influenza virus kills 250,000-500,000 people every year

Structure of Influenza Neuraminidase

The Greatest Known Influenza Pandemic the Spanish Flu of 1918-1920

-rivals the Black Death (Bubonic/Pneumonic) Plague of 1300s for deaths
-most of the victims young healthy adults
(like the current H1N1 pandemic) 2009
-1/3 world population (1.6 billion) infected
-50-100 million dead (3-6% of world population at that time)
-10 to 20% death rate
Country
India
U.S
UK
France
Canada

Deaths
17 million
675,000
250,000
400,000
50,000
comparisons:

64,000 Canadians died in WWI

45,000 Canadians died in WWII

Little could be done for victims

in 1918

except recommend gargling,

which would have close to no
positive effect

Dispatches from Fredericton circa 1918

Just about the time in 1918, when we thought the tragedy of death at war was
coming to an end, the Spanish influenza hit the world with millions of people
dying.
Many a soldier survived the horrors in the battlefields only to lose his life to this
flu either in the trenches or after returning home.
In October of 1918, Dr. William Roberts, the minister of health in New
Brunswick, outlawed the gathering of more than five people. Schools and
churches were closed for five weeks in an effort to combat the spread of the
Spanish influenza.
The illness would hit young adults with cold like symptoms that quickly led to
pneumonia. Often within a few hours the person was dead. Some people who
appeared healthy at bedtime were found as a corpse in bed in the morning others were healthy at dawn and dead by nightfall.
-from Victory Over Blindness Arthur Pearson

HIV protease inhibitors

Three main HIV enzymes:

Reverse Transcriptase
-copies viral genome
Integrase
-inserts viral genome into
host (human) chromosomes
Protease
-processes viral proteins so they
can assemble into new viruses

HIV protease structure

Many drugs (such as Saquinavir) available

for AIDS patients are protease inhibitors
(protease transition-state analogs)
http://www.youtube.com/watch?v=RO8MP3wMvqg

Electron Flow
The making and breaking of chemical bonds
Proteins are composed of amino acids (and often co-factors such as co-enzymes
and metal ions).

Amino acid side chains are the main determinants of protein structure and
function
Hydrophobic side-chains play major roles in overall structure of protein
hydrophobic effect van der Waals interactions
Polar (ionizable) side chains located in the active site of enzyme play a major role
in catalysis
Ionization involves proton loss or gain and about 75% of all reaction steps
catalyzed by enzymes involve the transfer of a proton

Seven amino acids have ionizable side chains

these are the amino acids with important roles in the active site
some play a direct role in catalysis and these are called CATALYTIC amino acids
others play an accessory role (eg. substrate/TS binding, other interactions) and
are just called active site amino acids

Roles of amino acid in active site

the phrases proton transfer and covalent binding imply CATALYTIC function

cation and anion binding or hydrogen bonding implies accessory roles in the active
site

Frequency of amino acids acting as catalytic residues

pKa values for

ionizable groups
on isolated amino acids

Notice that environment can

alter pKa values of amino acids

pKa values for

ionizable groups
on amino acids in
proteins
aspartate, glutamate
histidine
cysteine
tyrosine
lysine
arginine
serine

the imidazole ring of Histidine

Note Histidine:
High frequency as catalytic amino acid
pKa around 6-7
physiological pH is about 6.8-7.4
Remember that when pH = pKa of an ionizable group, both the protonated form
and the unprotonated form have the same abundance
(What equation could you use to demonstrate this ??)
Recall that most chemical reactions involve the transfer of protons.
This is why Histidine is such a common catalytic residuea pKa poised at the
physiological pH means that it can readily accept or donate protons during
chemical reactions occurring in the cell.

Next day we will take a closer look at the chemical reactions catalyzed
by enzymes and the roles played by amino acid side chains in the
active site.