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Enzyme Catalysis I

Formation of the Transition State

Great White Shark (Carcharodon carcharias)

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Enzyme-Substrate Binding

• enzyme catalysis begins with substrate binding to enzyme

• 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

E + S ES E + Pk 

-1 

k 1 

  k 2 

fast slow

E + S ES E + Pk 

-1 

k 1 

  k 2 

fast slow

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The Proximity Effect

• consider these non-enzyme catalyzed reactions

1.

2.

3.

4.

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Effect of Substrate Binding and Stabilization of Transition State

on energy profile of reaction

note that activation energy is lowered in two ways

b l

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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

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consider the reaction:

bromomethane + hydroxide anions → methanol and bromine anions 

• 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

bond beingformed

bond beingbroken

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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

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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 whenbound 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)

 

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

in time.

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open conformation closed conformation

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

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Cholesterol lowering drugs

Transition state analogs are often potent enzyme inhibitors and have various

therapeutic uses

Statins (like Lipitor) arecholesterol lowering drugs

because they are transition-

state analog inhibitors of

an enzyme called HMG-CoA

Reductase, a key enzyme in

the biosynthetic pathway forcholesterol.

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Antiviral DrugsTamiflu - Influenza Neuraminidase Inhibitor

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

H1N1

H1N5

etc

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Structure of Influenza Neuraminidase

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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)

-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 Deaths

India 17 million

U.S 675,000UK 250,000

France 400,000

Canada 50,000

comparisons: 64,000 Canadians died in WWI

45,000 Canadians died in WWII

2009

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Little could be done for victims

in 1918

…except recommend gargling, 

which would have close to no

positive effect

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“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 peopledying.”

“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 NewBrunswick, 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 whoappeared 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 

Dispatches from Fredericton – circa 1918

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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

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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

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Electron Flow

The making and breaking of chemical bonds

• Proteins are composed of amino acids (and often co-factors such as co-enzymesand 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

S i id h i i bl id h i

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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

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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

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pKa values for

ionizable groups

on isolated amino acids

pKa values for

ionizable groups

on amino acids in

proteins

aspartate, glutamatehistidine

cysteine

tyrosine

lysine

arginine

serine

Notice that environment can

alter pKa values of amino acids

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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 residue…a pKa poised at the

physiological pH means that it can readily accept or donate protons during

chemical reactions occurring in the cell.

the imidazole ring of Histidine

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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.