BIO 462

ENZYME AND ENZYME KINETICS

By:
REENA RASHID
SEPT 2015
 Learning Outcomes
• Explain enzyme, substrate and enzyme specificity
• Able to explain factors affecting rate of enzymatic
reaction
• Understand the function of the non-protein
substance in enzymatic reaction and give
examples
• Understand the enzyme kinetics invoving
Michaelis-Menten equation and Lineweaver-
Burke plot
Enzymes are effective Biological Catalyst
• Most efficient catalysts known
– Can increase reaction by a factor up to 10²⁰
• Non-enzymatic catalyst can enhance the rate of
reaction by factors of 10² to 10⁴
• All enzyme (except ribozymes) are globular
proteins
• Speeds up reaction but does not alter equilibrium
constant
• Reaction rate depends on the free activation
energy
Rate of reaction
• Transition state
– A state where the right amount of energy needed
to produce product
– Least stable state

• Reaction with or without enzyme:

– The standard free energy remain unchanged
– the activation energy is lowered, ΔG
 Coenzymes and Cofactors
• Non protein substance sometimes needed for enzymatic
catalysis

• Coenzymes are organic molecules that are required by

certain enzymes to carry out catalysis.

• They bind to the active site of the enzyme and participate

in the catalysis
– but are not considered substrates of the reaction.

• Coenzymes function as intermediate carriers of electrons,

specific atoms or functional groups that are transferred in
the overall reaction.
 coenzyme abbreviation entity transfered
nicotine adenine NAD - partly composed electron
dinucelotide of niacin (hydrogen atom)

nicotine adenine NADP -Partly composed electron

dinucelotide phosphate of niacin (hydrogen atom)

flavine adenine FAD - Partly composed electron

dinucelotide of riboflavin (vit. B2) (hydrogen atom)

coenzyme A CoA Acyl groups

electrons
coenzymeQ CoQ
(hydrogen atom)
thiamine pyrophosphate thiamine (vit. B1) aldehydes

pyridoxal phosphate pyridoxine (vit B6) amino groups

biotin biotin carbon dioxide

carbamide coenzymes vit. B12 alkyl groups

• Cofactors are often classified as inorganic
substances that are required for, or increase
the rate of, catalysis.
Cofactor Enzyme or protein
Zn2+ carbonic anhydrase
Zn2+ alcohol dehydrogenase

Fe3+ or Fe2+ cytochromes, hemoglobin

Fe3+ or Fe2+ Ferredoxin

Cu2+ or Cu+ cytochrome oxidase

K+ and Mg2+ pyruvate phosphokinase

 Condition for enzymes reaction
Temperature
• Enzymes are highly sensitive to changes in temperature.
– In humans, most enzymes function best at body temperature
(approximately 37oC).

• In general, heating a reaction mixture speeds up the rate of

reaction by increasing the number of successful collisions
between reactant molecules.

• If the temperature of the reaction is raised too high past

the optimum temperature, the reaction rate decreases as
the enzyme becomes denatured and loses its ability to
function as a catalyst for the reaction.
pH
• Some enzymes can only act within a certain pH
range
• Outside this pH range the enzyme may become
denatured, lose its tertiary structure, and
therefore lose its ability to function as a catalyst
for the reaction
Substrate Concentration
• Vmax reflects how fast the enzyme can catalyze the
reaction
• Based on Michaelis-Menten research on enzyme
kinetics study
 Will the reaction goes faster if you
raise the temperature ?
• A reaction will go faster at ↑ temperature
• However, if its catalyzed by enzyme, the
temperature can only be increased at a
specific range
• If the temperature is too high
– Enzyme denature and rate of reaction will be
reduce significantly
 Enzyme-Substrate Binding
• Enzymes bind to substrate forming a ES complex

• Substrates bind non-covalently at the active site

– Highly specific interactions
– An attraction between E and S must exist for them to bind
– An enzyme catalyzed reaction may be written as:
E + S ↔ ES ↔ E + P

• Two important models hat describe the interaction

Lock and Key Induce Fit

Model Model
 Lock and Key
• The active site have a fixed structure (the lock),
which exactly matched the structure of a specific
substrate (the key).
– Like a correct jigsaw puzzle
 Induced fit model
• The bindings site has a different 3 D shape before
it bind to substrate
• Binding of the substrate induces conformational
change in the active site that results in
complementary fit
 Michaelis-Menten Approach on Enzyme
Kinetics
• Initial rate of reaction V ini or V0
• As the rate increases, it would depend on
concentration
• Until at one point, all the active sites of an
enzyme are saturated, the reaction is at Vmax
 Vmax [S] Michaelis-Menten
Vinit =
KM + [S] equation

 it is difficult to determine Vmax experimentally from a

hyperbola graph plot
 The value would never reached any finite substance in lab
experiment

 So how do we calculate the Vmax from graph?

 Lineweaver-Burk Plot
• Use Lineweaver-Burk Plot double-reciprocal
plot to calculate Km and Vmax from a straight
line graph
• a plot of 1/V versus 1/[S] will give a straight line
with slope of KM/Vmax and y intercept of 1/Vmax
• KM is the dissociation constant for ES

KM
slope =
Vmax
1
x intercept = -1 V
KM

y intercept = 1
Vmax

1
[S]
 Enzyme Inhibition

• An inhibitor-substance interferes with the action of

enzyme and slows the reaction
• How?
– Irreversible inhibitor
• Reacts with enzyme and inactivates it by modifying the original
enzyme

– Reversible inhibitors
• Inhibitor bind to the enzyme →inhibits binding to substrate →
inhibitor released enzyme → enzyme back in its original
condition
• Has 2 types:
– Competitive inhibition
– Non-competitive inhibition