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Catalysts in biochemical reactions
1) DEFINATION:
WHAT IS ENZYME?
Enzyme is a class of proteins that function as catalysts in biochemical
reactions. On their Characteristics is that they increases the rate of reactions
by several orders of magnitude A very dramatic example of enzyme kinetics is
given by decomposition of hydrogen
Peroxide. Enzymes are usually protein molecules that manipulate other
molecules — the enzymes' substrates. These target molecules bind to an
enzyme's active site and are transformed into products through a series of
steps known as the enzymatic mechanism. These mechanisms can be
divided into single-substrate and multiple-substrate mechanisms. Kinetic
studies on enzymes that only bind one substrate, such as triosephosphate
isomerase's, aim to measure the affinity with which the enzyme binds this
substrate and the turnover rate.
When enzymes bind multiple substrates, such as dihydrofolate reductase
(shown right), enzyme kinetics can also show the sequence in which these
substrates bind and the sequence in which products are released. An
example of enzymes that bind a single substrate and release multiple
products are proteases, which cleave one protein substrate into two
polypeptide products. Others join two substrates together, such as DNA
polymerase linking a nucleotide to DNA. Although these mechanisms are
often a complex series of steps, there is typically one rate-determining step
that determines the
Overall kinetics
GENERAL PRINCIPLE OF ENZYME KINETICS:
Reaction rates increase as substrate concentration increase, but become
saturated at very high concentrations of substrate.The reaction catalyzed by
an enzyme uses exactly the same reactants and produces exactly the same
products as the unanalyzed reaction. Like other catalysts, enzymes do not
alter the position of equilibrium between substrates and products. However,
unlike uncatalysed chemical reactions, enzyme-catalysed reactions display
saturation kinetics. For a given enzyme concentration and for relatively low
substrate concentrations, the reaction rate increases linearly with substrate
concentration; the enzyme molecules are largely free to catalyze the reaction,
and increasing substrate concentration means an increasing rate at which the
enzyme and substrate molecules encounter one another. However, at
relatively high substrate concentrations, the reaction
rate asymptotically approaches the theoretical maximum; the enzyme active
sites are
almost all occupied and the reaction rate is determined by the intrinsic
turnover rate of the enzyme. The substrate concentration midway between
these two limiting cases is denoted by KM.[2]
HOW DOES ENZYME WORK?
1.Enzyme-catalyzed reactions are characterized by the
formation of a complex between the enzyme and its substrate
(the ES complex).
2.Substrate binding occurs in a pocket on the enzyme called
the active site. Enzymes accelerate reactions by lowering the
remains unaffected by the enzyme do this by binding the
transition state of the reaction better than the substrate….[3]
METHODOLOGY:
The rate of reaction is estimated to be 1/100000000 /m /s in the absence of a
catalyst .this could very well be the upper limit since one can never be certain
of avoiding the catalytic action of dust particles. In the presence of catalyses
an enzyme found in the never and other organs, the rate of the reaction is
10000000/m/s
Another important characteristic of enzyme kinetics is specifilty.for example,
enzyme urease catalysis the decomposition of urea to ammonia and carbon
dioxide but it found to been effective in other reaction. Enzyme is a class of
reaction, and their name usual reflect this aspect of the chemistry………..
Particular classes of reaction common in biochemistry have the form:
S-------enzyme-------------------------àP
Where a compound s (usually called the substate) is transformed to a product
p, under the
Influence of an Enzyme. Since an enzyme is specific to a class of substrates,
it is responsible to assume that a complex is formed between the enzyme E
and substate: 4)
E+S -------àES (COMPLEX)
MICHAELIS-MENTEN EQUATION:
In 1913 L.Michalis and M.L.Menten proposed a mechanism of enzyme action
that involves equllibirum among the enzyme ,the substate , and the complex
ES.G.E .Briggs and J.B.S Haaldane showed that the michaelis -mentaen
mechanism with less restrictive steady state hypothesis for ES ,leads to a
correct rate law .Consist of an enzyme binding to a substate to form a
complex which dissocites to give either the product or the substate:
(RUN ENZYME KINETICS)
The kinetics of single-substrate enzyme-catalysis. The interactions between
enzymes and substrates are often difficult to understand and the model
allows users to visualize the complex reaction.
The standard equation for this reaction is shown below:
Kc Kr
E + S <=======> E-S ------> + P
Kd
At the concentration of P is zero ,and thus there is no back reaction.the intial
rate of formation of the product is there given by :
d[p]
vo = ------ = k3 [ES]
dt
According to steay state approximation,
D [ES]
------- = 0 = - (k2+k3)[ES]+k1[E][S]
dt
Michaelis-Menten equation the conc. Of enzyme at a given time is related to
this intial conc. [E]o by
[E] = [E] - [ES]
(Free) (Intial) (Bound)
This assumption implies that each enzyme molecule provides one side in
binding of the substate ……
[ES] = K1[E]o[S]
---------------K2+ K3+ K1[S]
From steady state conc of ES:
VO = K3K1 [E]o[S]
-----------------K2+K3+ K1[S]
For intial rate .this equation is frequently written as :
Vo = k3[E]o[S]
------------Km+ [S]
Where
Km = k3+ k2
---------K1
Is called the Michaelis constant.
Let us consider the case of [S]>Km
Vo =k3 [E]o = vm (2)
The intial concetation of enzyme is also its maximum concentration .hence
the intial velocity with a large substate conc. must be the max. Velocity Vm.
The reaction is zero order reaction in substreateconc. This property of
enzyme was first observed by A.Brown in 1903 , while studying the enzymatic
hydrolysis of sucrose. The rate substate molclules converted is called
turnover number because it is equal to no of substrates.[4]
TURNOVER NUMBER:
The turnover no for catalase, which catalyzes the decomposition of hydrolysis
peroxide is 5.. The volume of oxygen generated during 1 s when 0.10g of
catalyse is added to excess hydrogen peroxide.the reaction peroxide
isothermally at 300K . The molar mass of catalyse is 60,000 Daltons
Hence
d[O2]
------- = 0.5 k3[E]o
dt
=4.2mol/s
This corresponds to 100 L of O2 per second![5]
The bombardier bettle uses this reaction effectively dense it carries a 25%
solution of
hydrogen peroxide in a sack and when threatened triggers the above reaction
…the
Sudden release of oxygen rapidly heats to a high temp ,making it possible for
bettle to
Spray the enemy with near boiling water.[6]
At low concentration [S] <Km and we have
Vo = Vm
-------Km
This indicates that the reaction is first order reaction in substrate show how
the velocity
of the reaction changes as a function of substate concentration. The order of
the reaction
changes from one to zero as the substrate concentration increases If k3<k2
the Michalies
Binding….the largest Michaels constant, the smaller the velocity constant is
measure
The equllibirum constant for enzyme substrate bindings. the large the
Michelins constant,. the smaller the velocity…..[7]
Variation in the intial rate of enzyme kinetics reaction as a function substrate
concentration:
Significance of KM
When [S] = KM, then V=Vmax/2. Hence KM is equal to the substrate
concentration at which the reaction rate is half its maximum value. In other
words, if an enzyme has a small value of KM, it achieves its maximum
catalytic efficiency at low substrate concentrations. Hence, the smaller the
value of KM, the more efficient is the catalyst. The value of KM for an enzyme
depends on the particular substrate. It also depends on the pH of the solution
and the temperature at which the reaction is carried out. For most enzymes
KM lies between 10-1 and 10-7 M.[7]
Determining KM and Vmax experimentally
To characterize an enzyme-catalyzed reaction KM and Vmax need to be
determined. The way this is done experimentally is to measure the rate of
catalysis (reaction velocity) for different substrate concentrations. In other
words, determine V at different values of [S]. Then plotting 1/V vs. 1/[S] we
should obtain a straight line described by equation (18). From the y-intercept
and the slope, the values of KM and Vmax can be determined. For example,
use EXCEL to plot the data shown below. Fit the data to a straight line, and
from the equation of the straight line determine the values of KM and
Vmax.[8]
LINEWEAVER -BURK PLOT:
The ES Complex break then to release the product and free enzyme:
E+ S………K1……>ES…K3……>.P+E
The rate is given by:
D[ES]/dt = k1([E]-[ES])[S]
-d[ES]/dt = k2[ES] + K3[ES]
At equallibrum two rates rep by th above equation
K1([E] - [ES] = K2 [ES] + K3[ES]
Rearrange the equation:
[S][E] -[ES] K2+K3
……………. = ……….. = Km
[ES] K1
Km is called Michaelis constant.
[ES] == [E][S]
……….
Km + S
Since intial rate v of enzyme is proposal to ES
V == k3[ES]
Vmax == k3 [E]
We can written also:
V = k3 = [E][S]
………..
KM + [S]
IN this reaction to use the final reaction is:
V = Vmax [S]
……….. [9]
Km + [S]
LINEWEAVER -BURK PLOT
ENZYME KINETICS MECHANISMS:
Ternary-complex mechanisms:
Random-order ternary-complex mechanism for an enzyme reaction. The
reaction path is shown as a line and enzyme intermediates containing
substrates A and B or products P and Q are written below the line.
In these enzymes, both substrates bind to the enzyme at the same time to
produce an EAB ternary complex. The order of binding can either be random
(in a random mechanism) or substrates have to bind in a particular sequence
(in an ordered mechanism). When a set of v by [S] curves (fixed A, varying B)
from an enzyme with a ternary-complex mechanism are plotted in a Line
weaver-Burk plot the set of lines produced will intersect.
Enzymes with ternary-complex mechanisms include glutathione S-transfer
dihydrofolate reductase and DNA polymerase The following links show short
animations of the ternary-complex mechanisms of the enzymes dihydrofolate
reductase and DNA polymerase.
2) Ping-pong mechanisms
Ping-pong mechanism for an enzyme reaction. Intermediates contain
substrates A and B or products P and Q.
As shown on the right, enzymes with a ping-pong mechanism can exist in two
states, E and a chemically modified form of the enzyme E*; this modified
enzyme is known as an intermediate.. In such mechanisms, substrate A
binds, changes the enzyme to E* by, for example, transferring a chemical
group to the active site, and is then released. Only after the first substrate is
released can substrate B bind and react with the modified enzyme,
regenerating the unmodified E form. When a set of v by [S] curves (fixed A,
varying B) from an enzyme with a ping-pong mechanism are plotted in a
Lineweaver-Burk plot, a set of parallel lines will be produced.[13]
Enzymes with ping-pong mechanisms include some oxidoreductases such as
thioredoxin peroxides, transferases such as acylneuraminate
cytydilyltransferase and serine proteases such as trypsin and chymotrypsin
Serine proteases are a very common and diverse family of enzymes,
including digestive enzymes (trypsin, chymotrypsin, and elastase), several
enzymes of the blood clotting cascade and many others. In these serine
proteases, the E* intermediate is an acyl-enzyme species formed by the
attack of an active site serine residue on a peptide bond in a protein
substrate.[14]
3) RNA as an Enzyme:
Although enzymes are considered to be proteins, enzyme activity has
recently been found in ribonucleic acid (RNA) in certain organisms. These
"ribozymes" may yield clues to the origins of life on Earth. DNA needs
enzymes to replicate, whereas enzymes need the instructions of DNA. This
represents a "chicken-and-egg" question that has stumped researchers. Early
life may have used RNA that was able to catalyze its own replication.
CONCLUSION:
Since enzymatic reactions are so important to biological chemical reactions, it
is of great interest to be able to model them. By use of the study of chemical
kinetics, it is possible derive rate equations for the steps involved in an
enzymatic reaction. These rate equations are differential equations and can
be used to model the concentrations of each compound in the system.
However, this system of differential equations is hard to determine
experimentally because of the difficulty of determining the rate constants. By
use of the Quasi-Steady-State Assumption, we can turn our system of
differential equations into the Michaelis-Menten enzyme equation. Many
benefits stem from this transition. One benefit is the fact that it is now easy to
determine the constants related to the enzyme equations. However, how do
we know the Quasi-Steady-State Assumption is valid? It seems reasonable
from a physical argument. By use of dimensional analysis, we can give a
more rigorous mathematical argument for the Quasi-Steady- State
Assumption. The Michaelis-Menten enzyme equation is very important in the
study of cellular