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The process of increasing the rate of reaction with the use of a catalyst.
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Enzymes
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http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg
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Enzymes
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Amylase
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Factors that may affect catalysis rates
Temperature
pH
Enzyme concentration
Amount of substrate
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Materials and Methods
Solution Preparation
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Estimation of salivary amylase activity
A drop from the digestion mixture mixed with 1 drop of iodine for every
minute.
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Effect of enzyme concentration
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Effect of amount of substrate
Six percent starch prepared from which five other dilutions were
prepared: 5%, 4%, 3%, 2%, and 1%.
The same procedure for Estimation of salivary amylase activity used
using 2.5% salivary amylase solution.
Reaction rates for each substrate dilution recorded.
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Effect of pH
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Effect of temperature
Test tube with 2.5% salivary enzyme was placed on water baths
maintained at 4oC, 10oC, 38oC, 58oC, 78oC and 100oC.
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Results and Discussion
Estimation of amylase activity
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Estimation of amylase activity
Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Saliva and
Their Corresponding Time to Reach the Achromic Points, Amylase Units and Enzyme Activity
Effect of Enzyme Concentration
0.5 30
0.833333333 0.4000
0.75 23
1.630434783 0.2667
1 18
2.777777778 0.2000
1.5 11
6.818181818 0.1333
2 7
14.28571429 0.1000
2.5 7
17.85714286 0.0800
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Effect of Enzyme Concentration
Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Saliva and
Their Corresponding Time to Reach the Achromic Points, Amylase Units and Enzyme Activity
Effect of Enzyme Concentration
Salivary Amylase time to achromic point (min) amylase units Enzyme activity
(%)
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Effect of Enzyme Concentration
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Effect of the Amount of Substrate
Rate of Reaction
– describes how fast a chemical reaction proceeds
– depends on reactant and product concentrations
• More importantly on rate constant k
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Effect of the Amount of Substrate
Enzyme Kinetics
– still follows the same trend
• Increasing either substrate or enzyme increases rate
– but there is a limit to this relation
• When enzyme conc are constant, there is a limit to the velocity of the
reaction
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Effect of the Amount of Substrate
Michaelis-Menten Kinetics
– enzymatic reactions are observed to reach a maximum rate of reaction Vmax
– constant enzyme concentration provides a limiting effect
• All enzymes are bound to substrate
– vo= Vmax [S] / (Km + [S])
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Effect of the Amount of Substrate
Rectangular Hyperbola
Max rate of Vmax
Half-Velocity is reached
at Km
Vmax is dependent on
[E}
Km is constant
– Km=K-1 + K2 / K+1.
– Measure of affinity
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Rate of Reaction vs. Substrate Concentration
Rate increases with substrate concentration
However exponential relation
Due to experiment limit (30 min)
Km and Vmax not evident
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Rate of Reaction vs. Substrate Concentration
Allowing time to go beyond 30, rectangular hyperbola is attained
Change in rate diminishes as substrate conc increases
Vmax still indiscernible together with Km
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Effect of the Amount of Substrate
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Effect of the Amount of Substrate
Linear equation
Regression Analysis
allows determination of
Km and Vmax
Also able to determine
nature of protein function
inhibition (competitive,
uncompetitive,
noncompetitive)
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Double Reciprocal Plot
Not strong liinear relation (due to experimental limits)
R2 value of only 0.785
Vmax= 4.24719E-05 Km= 0.000484646
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Double Reciprocal Plot
Perfect linear relation
Vmax= 2.08333E-05 Km=4.16667E-05
Very Low Km, high affinity of enzyme
Vmax close to velocity values, near saturation
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Effect of pH
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Effect of pH
• Enzymes
– have a range of pH at which it is active and outside of which it is inert
– optimum pH
• most favorable pH value
• point where the enzyme is most active
• extremely high or low pH values generally result in complete loss of activity
for most enzymes
• Salivary amylase has an optimum pH of around 7
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Effect of pH
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Effect of Temperature
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Effect of Temperature
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Effect of Temperature
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Effect of Temperature
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Conclusion
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