Transcript ENZYMES (Basic Concepts and Kinetics) (Chapter 8)

ENZYMES
ENZYMES
•are biological catalyst
•are mostly proteinaceous in nature,
but RNA was an early biocatalyst
•are powerful and highly specific
catalysts
carbonic anhydrase
Many enzymes require
co-factor for activity
Apoenzyme + co-factor = holoenzyme
NOMENCLATURE:
1. Common name- e.g. trypsin, pepsin
2. Hybrid name- e.g. sucrase
3. Systematic name: EC 2.7.4.4
example: nucleoside monophosphate kinase
ATP + NMP  ADP + NDP
ENZYMES accelerate
reactions by facilitating the
formation of the transition
state
Active site
• is the region where the substrate binds
• contains residue that directly participate in making
or breaking of bonds (formation of transition state)
• is the region where activation energy is lowered
Common features
1. Active site is a three dimensional cleft
2. Takes up a small part of the total volume of an enzyme
3. Are clefts or crevice
4. Substrates are bound to enzymes by multiple weak
interactions
Two models of the Active site
1.Lock and key
2.Induced-fit
Kinetic Properties of Enzymes
Michaelis-Menten Equation
Factors Affecting Enzyme Activity
1. Temperature
2. pH
3. [S]
4. Presence of Inhibitors
TEMPERATURE
•As the temperature rises, molecular
motion - and hence collisions between
enzyme and substrate - speed up. But
as enzymes are proteins, there is an
upper limit beyond which the enzyme
becomes denatured and ineffective.
pH
•The conformation of a protein is
influenced by pH and as enzyme
activity is crucially dependent on its
conformation, its activity is likewise
affected.
Kinetic Theory of EnzymeCatalyzed Reaction
1. Effect of [E]
 involves the reversible formation of an
enzyme-substrate complex, which then break
down to form one or more products
 if [S] is constant, v is proportional to [E]
2. Effect of [S]
 has profound effect on the rate of enzymecatalyzed reaction
o
At low [S], rate of reaction is 1 order,
v is directly proportional to [S]
At mid [S], rate of reaction is mixed order
proportionality is changing
At high [S], rate of reaction is zero order
Michaelis-Menten Equation
Significance of KM
When V= ½ Vmax, what is [S]?
The KM of an enzyme is the
substrate concentration at which
the reaction occurs at half of the
maximum rate.
There are limitations in the
quantitative (i.e. numerical)
interpretation of this type of
graph, known as a Michaelis
plot. The Vmax is never really
reached and therefore Vmax and
hence KM values calculated from
this graph are somewhat
approximate.
Lineweaver- Burk plot
The Effects of Enzyme Inhibitors
1. Competitive
In the presence of a competitive inhibitor, it takes a higher substrate
concentration to achieve the same velocities that were reached in its
absence. So while Vmax can still be reached if sufficient substrate is
available, one-half Vmax requires a higher [S] than before and thus Km
is larger
2. Non-Competitive
With noncompetitive inhibition, enzyme molecules that have been
bound by the inhibitor are taken out of the game so
•
enzyme rate (velocity) is reduced for all values of [S], including
•
Vmax and one-half Vmax but
•
Km remains unchanged because the active site of those enzyme
molecules that have not been inhibited is unchanged.
Most Biochemical Reactions Include Multiple
Substrates:
A+BP+Q
2 Classes of Multiple Substrate Reactions:
1. Sequential Displacement
2. Double Displacement
Sequential Displacement:
All substrates bind to the enzyme before any product
is release
Types
• Ordered
• Random
Example of a sequential ordered mechanism
Lactate dehydrogenase
The enzyme exist as a ternary complex
Example of a Random Sequential Mechanism
Creatine kinase
Double Displacement (Ping-Pong) Reactions
-one or more products are released before all
substrates bind the the enzyme
- a substituted enzyme intermediate exist
Aspartate amino transferase
Enzymes employ strategies to catalyze
specific reactions
1.Covalent Catalysis- the active site
contains a reactive group
2.General Acid base catalysis
3.Metal ion catalysis
4.Catalysis by approximation
1. Covalent Catalysis
Example: Chymotrypsin
A. Acylation to form the acyl-enzyme intermediate
B. Deacylation to regenerate the free enzyme
2. General Acid base catalysis
3. Metal ion catalysis- e.g. carbonic anhydrase
Regulatory Strategies:
1. Allosteric Enzyme- e.g. ATCase
2. Multiple of Enzymes:
Isoezymes or Isozymes- are homologous
enzymes within a single organism that catalyze
the same reaction but differ slightly in structure
and in Vmax and Km
e.g. Lactate dehydrogenase (LDH)- 2 isozmic
chains in humans, H (heart) and M (muscles)
3. Reversible covalent modification
4. Proteolytic activation- involves synthesis of
enzymes in the ZYMOGEN form
Examples:
1. Digestive enzymes
2. Blood clottingcascade of
zymogen
activations