Enzymes

Enzyme Kinetics & Enzyme Inhibition

 Enzymes = Biological catalysts
Permit reactions
Large proteins to ‘go’ at
body conditions
pH 7.4, 37oC
Process millions
of molecules
every second
Very specific
react with only
1 or a few types
of molecules
(substrates).
 Effect of enzymes on Eact

For all reactions

you must get over
the activation
energy hurdle.
Ea
Reactants
Energy

H2O2

H Products
H2O + O2
 Effect of enzymes on Eact
Enzyme catalyzed Enzymes change
reaction how reactions
proceed.
Reducing
Ea activation energy.
Reactants
Energy

H2O2 Makes faster.

H Products
H2O + O2
Enzyme nomenclature
• Name is based on:
what with or
how
it reacts
+ -ase
ending
Examples
To react with lactose.
lactase
To remove carboxyl from pyruvate.
pyruvate decarboxylase
Classification of enzymes
• Based on type of reaction

• Oxireductasecatalyze a redox reaction

• Transferase transfer a functional group
• Hydrolase catalyze hydrolysis rxns
• Lyase Add or remove to C=C bonds
rearrange to form isomers
• Isomerases
join two molecules
• Ligase
The Active Site
• Enzymes are typically HUGE proteins, yet only a small
part are actually involved in reaction.

The active site has two

basic components.
catalytic site
binding site
Model of
trios-p-isomerase
The Active Site

Catalytic site
Binding Where reaction
occurs
Site
holds
substrate
in
place Substrate

Enzyme
SPECIFICITY
• Enzymes are very specific. Each enzyme will catalyze
only one type of reactions and often will only work with
a specific substrate.
•
• Ex. NH2-C-NH2 + H20 2NH 3 + CO2
urease

O
• Urease has no effect on other compounds.
• Such absolute specificity is rather rare among enzymes.
Enzyme classes
• Absolutely specific
• Only reacts with a single substrate.

• Group specific
• Works with similar molecules with the same functional group.

• Linkage specific
• Catalyzes a specific combination of bonds.

• Stereochemically specific
• Only will work with the proper D- or L- form.
• ISOENZYMES: Different enzymes that perform the same type of
function in different organisms or tissues.
Enzyme-substrate complex
• Step 1: (All of these steps are in equilibrium)
• Enzyme and substrate combine to form complex
•E + S ES
• Enzyme Substrate Complex

+
Enzyme-product
• Step 2: complex
• An enzyme-product complex is formed.

• ES EP

ES transition EP
state
Product
• The enzyme and product separate

• EP E + P
The product
is made
Enzyme is
ready
EP for
another
substrate.
Lock and Key Theory
•Enzyme is “lock” and Substrate is the “key”.
•Substrate structure
•must fit into enzyme’s structure.
Induced Fit Theory
•Active site may not fit substrate.

•Site must change in order to form the complex.

Effect of Temp on Enzymatic Rxns
• Exceeding normal pH and temperature ranges
always reduces enzyme reaction rates.

Optimum Temp
Reaction Rate

usually
37oC.

Temperature
 Effect of pH on Enzymatic Rxns

Most enzymes
work best near pH 7.4
Reaction Rate

not all though.

pH
Examples of optimum pH
• Optimum
• Enzyme Source pH

• pepsin gastric mucosa 1.5

• sucrase intestine 6.2
• catalase liver 7.3
• arginase beef liver 9.0
• alkaline bone 9.5
• phosphatase
Effect of substrate concentration
For non-enzyme catalyzed reactions
Rate of reaction
(velocity)

Rate increases if
concentration of
the substrate increases

Substrate concentration
 Effect of substrate concentration
For Enzyme catalyzed reactions

Rates increase but only to a certain point

Vmax w/ more enzyme Saturation point
Rate of reaction

Vmax w/ some enzyme At Vmax

(velocity)

the enzyme is working as

fast as it can.
Rate is limited by
the concentration of both
the substrate and enzyme.

Substrate concentration
 Effect of Enzyme concentration

Enzyme
Activity

Enzyme Concentration
Turnover Number
• Turnover Number:
• The rate at which an enzyme transforms the substrate
• Is measured at optimum pH and temperature.

Example:
Carbonic Anhydrase

H2CO3 H2O + CO2

36,000,000 molecules
minute
ENZYME
• InhibitorsINHIBITION
= interfere with ability of enzyme to react
properly with its substrate.

• For example:
– Medicinal drugs
• inhibit by inactivating an enzyme essential to bacterial growth.

• Viruses more difficult to inhibit because they use enzyme

system of the host cell.
• (An inhibitor of a virus also
• destroys host cells)
ENZYME INHIBITION
• Two Types of Inhibitors:
–Competitive

–Noncompetitive
COMPETITIVE INHIBITOR

Competes with
substrate for
the active site.

Enzyme
mistakes
inhibitor for
substrate
End product inhibition
Enzyme - substrate reactions in equilibrium.

• E+S ES ES* EP E+P

Equilibrium shifts left Inc P

If product builds up,

the reaction slows.
 Reversible Competitive inhibition
Enzyme - substrate reactions in equilibrium.

Inhibitor Substrate

EI ES

EI I + E + S ES EP  E + P
shifts Inc I Inc S Shifts
Competitive Inhibitors
Sulfa Drugs
• Illnesses caused by invading microorganisms like
bacterium can be combated using a competitive
inhibitor called an antimetabolite.

• Folic Acid is a coenzyme in many biosynthetic processes

like synthesis of amino acids and nucleotides.
 O
Sulfa Drugs C
OH
Folic Acid : obtained
• from the diet or H N
p-aminobenzoic acid
H
• from microorganisms in the intestinal tract.

Microorganisms make folic acid from PABA.

H2N N N
O O
N NH C NH CH C OH
N
CH2
OH
CH2
C OH
O
• In 1930 it was discovered that sulfanilamide, along with sulfapyridine
and sulfathiazole, could kill many types of harmful bacteria and help
cure several diseases.

• The bacteria are tricked into using the sulfa drugs instead of PABA.

• They make a molecule that also has a folic acid type of structure but is
not exactly the same.
• When they try to use this fake folic acid as a coenzyme, not
only doesn’t it work, it is now a competitive inhibitor.
• Many of the bacteria’s amino acids and nucleotides cannot
be made, and the bacteria die.
Penicillin: War of Enzyme against Enzyme.
• Produced by mold, it prevents growth of bacteria by successfully
competing for active sites on an enzyme that bacteria need for cell
wall production.
• 1. Bacteria need the enzyme transpeptidase to make
their cell walls rigid and cross-linked.
• 2. Penicillin takes control of transpeptidase.
• 3. Bacteria cell walls are not cross-linked and the contents
of the bacteria cells cannot be held in.
• 4. Cytoplasm spills out, and the bacteria die.
• Penicillin is an example of a beta-lactam.
• It has within its structure the beta-lactam ring.
• Over the past 15-20 years a new strain of bacteria has
developed that can resist penicillin. They contain an enzyme
(penicillinase) that opens the beta-lactam ring and renders
the penicillin ineffective.
By changing the R group, science has found a
way to prevent this from happening.
Non competitive Inhibition
• This type of inhibitor is believed to alter the shape of the enzyme and
greatly reduce its affinity for the substrate.
• 1. It does not compete with the substrate for the
active site.
• 2. It does not need to resemble the structure of the
substrate.
• 3. Its’ effect cannot be reversed by increasing the
substrate concentration.
 Non competitive Inhibition
•A noncompetitive inhibitor can bind to an
enzyme in many ways.
• If it binds somewhere on the surface of
the enzyme, it causes a change in the
tertiary structure.
•The substrate is inhibited because it can’t
get into the enzyme.