The Michaelis-Menten Model Accounts For the Kinetic Properties
of Many Enzymes
For many enzymes, the rate of
catalysis V0, defined as the
number of moles of product
formed per second and varies
with the substrate conc.
Vo=Vmax[S] /[S]+KM
Km is equal to the substrate concentration at which the reaction rate is half
maximal value.
The following data were recorded for the enzyme catalyzed reaction S
[S]
(M)
6.25 x 10-6
7.5 x 10-5
1.00 x 10-4
1.00 x 10-3
1.00 X 10-2
Michaelis-Menten Equation
P
V
(nmoles x liter-1 x min-1)
15.0
56.25
60
74.9
75
a. Estimate Vmax and Km. b what would be v at [S]=2.5 x 10-5M and [S]=5 x 10-5M c. what
would v be at if enzyme concentration is doubled?
The Km value for an enzyme depends on the particular substrate and environmental conditions
such as pH, temperature and ionic strength
1
Km and Vmax values can be determined by several means
Vmak can be accurately determined if the Michaelis-Menten equation is transformed into
One that gives straight-line plot . Taking the reciprocal of both side of equation:
1/V0= Km/Vmax*1/S+1Vmax
A plot of 1/Vo versus 1/S called a Lineweaver-Bruk or double-reciprocal plot
The Significance of Km and Vmax values
The Km value depends on the particular substrate and on environmental conditions
such as pH temperature, and ionic strength. The Michaelis constant has two meaning:
1. Km provides a measure of the substrate conc. required for significant catalysis
occurs.
2. Km is equal to the dissociation constant of the ES complex if k2 is much smaller than
k-1
kcat/Km is a measure of catalytic efficiency
When [S]>>>Km the rate of catalysis is equal to Vmax
Under the physiollogical condition [S]/Km ratio is typically between 0.01 and 1
The maximal rate reveals turnover number of an enzyme, which is the number of
molecules converted into product by an enzyme molecule in a unit time when the
enzyme is fully saturated with substrate (which is also called kcat).
When [S]<<<<Km the enzymatic reaction is much less than kcat
Vo=kcat/Km [E][S]
2
Most Biochemical Reactions Include Multiple Substrate
Most reactions in biological system usually include two substrate and two products.
Multiple substrate reactions can be divided into two classes: sequential displacement
and double displacement.
In the sequential mechanism, all substrates must bind to the enzyme before any
product is released. Sequential mechanism are of two types. Ordered and random.
3
Cleland notion
In the random sequential mechanism, the order of the addition of substartes and
release of products is random.
2. Double displacement (Ping-Pong) Reactions: One or more products are released
before all substrates bind enzymes.
4
Enzymes Can Be Inhibited by Specific Molecules
Inhibiting enzyme activity serves as a major control mechanisms in biological system.
Enzyme inhibition can be reversible and irreversible.
An irreversible inhibitor dissociates very slowly from its target enzyme because it has
become tightly bound to the enzyme, either covalently or non covalently. Example
penicillin binds to transpeptidase.
Reversible inhibition is characterized by a rapid dissociation of the enzyme-inhibitor
complex.
1.In the competitive inhibition an enzyme can bind substrate forming either ES or EI
cannot for ESI. therefore, a competitive inhibitor diminishes the rate of catalysis by
the reducing proportion of enzyme molecules bound the substrate.
2. In noncompetitive inhibition the inhibitor and substrate can bind simultaneously to
enzyme molecule at different binding sites.
Irreversible Inhibitors Can Be Used to Map Active Sites
Irreversible inhibitors can be divided into three categories: Group specific reagents,
substrate analogues, and suicide inhibitors.
Group specific reagents react with specific R groups of amino acids.
Example diisopropylphosphofluoridate (DIPF) modify only serine residue in active site
iodoacetamide can inactivate an enzyme by reacting with critical cysteine residue.
5
Substrate analogue (affinity labels) are the molecules that is structurally similar to
substrate for the enzyme that covalently modify active site residues.
Bromoacetol phosphate mimics natural substrate of TIM which is dihydroxyacetone
phosphate
Suicide inhibitors are modified substrate that provide the most specific means to modify
an enzyme active site.
Monamine oxsidase oxidize N,N-dimethylpropargylamine, which in turn inactivates the
enzymes by covalently modifying the flavin group.
6
Vitamins Are Often Precursors to Coenzyme
Monoamine oxsidase deaminates neurotransmitter such as dopamine and seretonin, lowering their
levels in their brain. Parkinson disease is associated with low levels of dopamine, and
depression is associated with low levels of seretonin.
Many enzymes require cofactors to be catalytically active. One class of these cofactors
termed coenzymes, consists of small organic molecules that are need in small amounts in diets
of some higher animal.
Vitamins can be grouped according to the whether they are soluble in water or in
nonpolar solvent.
Transition state analogs are potent inhibitors of enzymes.
7
8
Catalytic Strategies
Enzymes commonly employ one or more of the following strategies to catalyze specific
reactions.
1. Covalent catalysis: Active site of enzymes contains a reactive group , usually
powerful nucleophile that becomes temporarily covalently modified in the course of
catalysis. Example chymotrypsin
2. General acid-base catalysis: A molecule other than water plays the role of a proton
acceptor or donor. Example chymotrypsin
3.Metal ions catalysis: Metal ions involve during the catalysis as an electrophilic catalyst,
stabilizer of charge on substrate. NMP kinase
4. Catalysis by approximation: Bring two substrate together perform catalysis
Proteases
Protein turnover is an important process in living systems. Proteins that have served
their purposes must be degraded so that their amino acid can be used for protein
synthesis. Proteases cleave proteins by hydrolysis reaction- addition of a water
molecule to a peptide bond.
Chymotrypsin Possesses a Highly Reactive Serine Residue
Chymotrypsin cleaves peptide bonds selectively on the carboxylterminal site of the large hyrophobic
amino acid such as Phe, Tyr, Met
9
Chymotyrpsin is a good example of the use of covalent modification as a catalytic
strategy. the enzyme employs a powerful nucleophile to attack unreactive
carbonyl group of the substrate. This nucleophile becomes covalently attached to
the substrate briefly in the course of catalysis.
Chymotrypsin Action Proceeds in Two Steps Linked by a Covalent bound Intermediate
The initial phase of the reaction was examined by using stopped-flow method.
At the beginning of reaction, stopped flow method revealed that a “burst”
phase during which the colored product was produced rapidly. Product was then
produced more rapidly. Product was then produced more slowly as reaction reached the
steady state.
The two steps are explained by the reaction of the serine nucleophile with the substrate to form
covalently bound enzyme intermediate.
1st step is the formation acyl-enzyme intermediate by nucleophile attack of Ser-195 and
releasing the alcohol p-nitrophenol.
2nd step is the formation of carboxylic acid component of the substrate and regeneration
of enzyme.
These results suggested that two hydrolysis proceeds two steps.
10
The Catalytic triad converts serine 195 into potent nuclophile
The Catalytic triad converts serine 195 into potent nuclophile
His residue serves to position the serine side chain and to polarize its hydroxyl group. In doing so,
the residue act as a general base catalyst, a hydrogen ion acceptor
The nucleophile attack changes the geometry around this carbon atom from trigonal to tetrahedral
11
This tetrahedral intermediated than collapses to generate the acyl-enzyme. This step is facilitated by
the transfer of a proton from the positively charged his residue to amino group formed by cleavage
peptide.
amine group departs
amine roup repleaced by
water
12
Catalytic Triads Are Found in Other Hydrolytic Enzymes
Many other proteins have subsequently been found to contain catalytic triads similar
to that discovered in chymotrypsin such as trypsinş elastase. The sequence of these
proteins are approximately 40% identical.
13
Trypsin cleaves at the peptide bond after residues with long positively charged side
residue.
Elastase cleaves at the peptide bond after amino acids with small side chains (alanine
and serine)
Other enzymes that are not homologue of chymotrypsin have been found to contain
very similar active site.
Subtilisin from bacteria
Catalytic Triads Has Been Dissected by Site-Directed Mut.
Not all proteases utilize strategies based on activated Ser residue. Classes of proteins
have been discovered that employ three alternative approaches to peptide bond
hydrolysis. These classes are
1. cysteine proteases
2. asparatyl proteases
3. metalloproteases
14
Cysteine residues, activated by a histidine, plays the role of the nucleophile attack
that attack the peptide bond.
The aspartyl proteases have a pair of Asp in active center and they act together
to allow a water molecule to attack the peptide bond.
15
The active site of metalloproteases contains a bound metal ions, almost always Zn,
that activates a water molecule to act as a nucleophile to attack the peptide carbonyl
group.
16