3. Introduction
What are ENZYMES?
• Most diverse group of proteins
• Acts as biological catalysts
– cause or speed up chemical reactions by lowering the
activation energy (Ea)
• Important biological reactions catalyzed by
enzymes:
– Metabolism
– DNA synthesis
– RNA synthesis
– Protein synthesis
– Digestion
4. Introduction
What are ENZYMES?
• Active site
– pocket or cleft
– contains amino acid
side chains that
participate in substrate
binding and catalysis
• Substrate
– Reactant molecules
5. Introduction
Properties of Enzymes
• Large protein molecules
• Reusable (example: 2 H2O2 [catalase]→ 2 H2O + 1 O2)
• Remain unchanged
• VERY specific
• Operate at very high speeds
• Rate of reaction is dependent upon temperature,
pH, [E], and [S]
• Usually work best with 60°C
• Denaturation occurs at very high temperatures
6. Introduction
BINDING MODELS: LOCK & KEY MODEL
• Emil Fischer
• Enzyme specificity
• Substrate has a
complimentary shape to
the active site
• Substrate: key :: Enzyme:
lock
• Wrong key = no reaction
7. • a.k.a. “hand and glove
model”
• substrate induces a
slight change in the
shape of the active site
so that they both
compliments each
other’s shape.
BINDING MODELS: INDUCED FIT MODEL
9. SALIVARY AMYLASE
• Digestive enzyme responsible for catalyzing
starch molecules into simpler sugars (i.e.
maltose and isomaltose)
• Secreted by the salivary glands
• OPTIMUM TEMPERATURE: 37ᴼC
• OPTIMUM pH: 5.6 – 6.7
10. STARCH
• Mixture of two polysaccharides: amylose and
amylopectin
• White, tasteless, solid carbohydrate
• Converted to glucose via hydrolysis
• Detected by iodine (blue-black color)
12. OBJECTIVES:
• to observe the effect of different
temperatures and pH on the enzymatic
activity and specificity of the salivary amylase
• to determine the optimal temperature and pH
values of the salivary amylase
13. Materials
FOR A. EFFECT OF TEMPERATURE:
• Enzyme solution (1 mL saliva + 9 mL distilled H2O+ 30 mL 0.5% NaCl
• Buffered starch (1% starch in phosphate buffer pH 6.7)
• 0.001 M Iodine solution
• 2 Spot plates
• Test tubes
• Medicine droppers
• Constant Temperature bath (4, room temp, 37, 50, 60 and 70 °C)
14. Materials
FOR B. EFFECT OF PH:
• Enzyme solution (1 mL saliva + 9 mL distilled H2O+ 30 mL 0.5% NaCl
• 2% Unbuffered starch
• 0.001 M Iodine solution
• Acetate buffer solutions (pH 4 and 5)
• Phosphate buffer solutions (pH 6.7 and 8)
• Bicarbonate buffer (pH 10)
• 2 Spot plates
• Test tubes
• Medicine droppers
• Water bath set at 37°C
15. Methodology
A. Effect of Temperature
2 mL enzyme solution
In separate test tube, 2 mL of
buffer starch solution
• Incubate in 4°C ice bath for 10
minutes
• Immediately mix 2 mL enzyme
solution to 2 mL buffer starch
solution.
Enzyme- starch
solution
• Immediately take 3 drops and put in
spot plate well.
• Add 2 drops Iodine solution.
16. • Continue incubation for 1 minute
• Quickly take 3 drops of enzyme solution
and 2 drops of iodine and add it to the
second spot plate well.
• Record color
• Label as “1 minute”
• Continue incubation for 1 minute
until a light yellow- colored
solution is observed.
Light yellow colored
solution
• Repeat procedures for other temperatures (RMT, 37, 50, 60 and 70°C)
*Use hot water for high temperatures
• Plot the reciprocal of time (1.t, min -1) versus the temperature (T).
• Determine optimum temp. of Amylase
Blue-Violet colored
solution
• Note time (t)
17. B. Effect of PH
1 mL 2% unbuffered starch
With 1 mL acetate buffer
(pH4)
2 mL enzyme solution
• Incubate for 10 minutes in 37° C
• Immediately take 3 drops and put in
spot plate well.
• Add 2 drops Iodine solution.
• Label as “zero minute”
• Record color
18. Blue-Violet colored
solution
Light yellow colored
solution
• Continue incubation for 1 minute
• Quickly take 3 drops of enzyme solution
and 2 drops of iodine and add it to the
second spot plate well.
• Record color
• Label as “1 minute”
• Continue incubation for 1 minute
until a light yellow- colored
solution is observed.
• Note time (t)
• Repeat procedures for the other pH (5, 6.7, 8, and 10) using the
appropriate buffer.
• Plot the reciprocal of time (1/t, min -1) versus the buffer pH (pH).
• Determine optimum pH of Amylase
25. Salivary Amylase
• Alpha amylase
– Form produced by your salivary gland and pancreas
• Hydrolyzes alpha bonds of large, alpha linked oligosaccharides
and polysaccharides
– Acts on linear alpha(1,4) glycosidic linkages
Starch + amylase maltose + salivary amylase + dextrin
27. Effect of Temperature
• Enzymes work best at a certain temperature
(optimum temperature) where the reaction rate is
at maximum
• Optimum temperature: 37 °C (body temperature)
• Lower than 37°C = slower reaction
• At 40 °C and above, enzymes start to denature
- Destruction of 2° and 3° structures
28. Effect of pH
• pH affects the detailed structure of the active
site of the enzyme
– Requires certain level of acidity and alkalinity
– Too much or too little H+ interferes with electric
charges and disrupts H-bonds
• Extremely high or low pH will result in the
complete loss of enzymatic activity
29. Effect of pH
• Optimum pH: pH 6.7 – 7
• pH 10 is when most enzymes are denatured
31. Conclusion:
Effect of Temperature on Enzymatic
Activity
• Enzymes work best at a certain temperature
(optimum temperature) where the reaction
rate is at maximum
• Optimum temperature = 37 °C
32. Conclusion:
Effect of pH on Enzymatic Activity
• Affects the ionization of acidic or basic amino
groups
• Affects the shape of the substrate
• Optimum pH for salivary amylase = pH 6.7 -7
Editor's Notes
Large protein molecules
Reusable (example: 2 H2O2 [catalase]→ 1 O2)
Remain unchanged
VERY specific (acts only on specific substrate)
-meaning there won’t be any (or at least very little) wasted by-product, thus the yield is usually high.
catalase can break down 2 million H2O2 per minute at 0oC!
As the name suggests, the substrate induces a slight change in the shape of the active site so that they both compliments each other’s shape. When the substrate-enzyme complex is formed the then product is formed, they would then have a different shape and are subsequently released from the enzyme. The reason the enzyme active site changes is due to the fact that enzymes are highly flexible, making them conformationally dynamic molecules.