Think about… 4.1 Metabolism 4.2 Properties and actions of enzymes 4.3 Factors affecting the rate of enzymatic reactions 4.4 Applications of enzymes Recall ‘Think about…’ Summary concept map

Want old, worn-out jeans? Stonewashed jeans Want old, worn-out jeans? I can help you!

Stonewashed jeans Wash jeans with stones. Stones break some cellulose fibres & remove the blue dye.

Now we can use enzymes to break down the fibres more quickly! Stonewashed jeans Now we can use enzymes to break down the fibres more quickly! enzyme

1 enzyme What are enzymes

2 enzyme What are the importance of enzymes to organisms

3 enzyme What are the advantages of using enzymes in the making of stonewashed jeans

different chemical reactions take place in living cells 4.1 Metabolism different chemical reactions take place in living cells respiration Important for protein synthesis keeping alive! lipid synthesis

4.1 Metabolism different chemical reactions take place in living cells sum of the chemical reactions that take place in an organism = metabolism (新陳代謝)

Metabolism Catabolism Anabolism 4.1 Metabolism Metabolism Catabolism (分解代謝) Anabolism (合成代謝)

1 Catabolism complex molecule simple molecules breaking-down reactions 4.1 Metabolism 1 Catabolism complex molecule simple molecules energy breaking-down reactions release of energy

1 Catabolism + example: respiration carbon dioxide water glucose 4.1 Metabolism 1 Catabolism example: respiration carbon dioxide water + glucose energy

Metabolism Catabolism Anabolism 4.1 Metabolism Metabolism Catabolism (分解代謝) Anabolism (合成代謝)

2 Anabolism simple molecules complex molecule building-up reactions 4.1 Metabolism 2 Anabolism simple molecules complex molecule energy building-up reactions requires energy

2 Anabolism example: condensation of glucose starch glucose energy 4.1 Metabolism 2 Anabolism example: condensation of glucose starch energy glucose

What are catabolism, anabolism and metabolism? 4.1 Metabolism What are catabolism, anabolism and metabolism? Catabolism all breaking-down reactions in an organism Anabolism all building-up reactions in an organism Metabolism all chemical reactions in an organism

What is the role of enzymes in metabolism? 4.2 Properties and actions of enzymes What is the role of enzymes in metabolism?

 reacting molecules product (生成物) energy barrier (能障) 4.2 Properties and actions of enzymes  reacting molecules product (生成物) energy barrier (能障)

Energy supplied to overcome the energy barrier energy barrier (能障) 4.2 Properties and actions of enzymes energy reacting molecules product (生成物) Energy supplied to overcome the energy barrier energy barrier (能障)

analogy: pushing a rock up a hill 4.2 Properties and actions of enzymes analogy: pushing a rock up a hill reacting molecule product

analogy: pushing a rock up a hill 4.2 Properties and actions of enzymes analogy: pushing a rock up a hill reacting molecule energy barrier product the reaction does not start due to the presence of energy barrier

analogy: pushing a rock up a hill 4.2 Properties and actions of enzymes analogy: pushing a rock up a hill maximum energy energy when energy is supplied … reaction starts

How can the energy barrier be overcome in our body? 4.2 Properties and actions of enzymes How can the energy barrier be overcome in our body?

smaller energy barrier 4.2 Properties and actions of enzymes By increasing the temperature? smaller energy barrier energy level raised energy barrier is easier to overcome

smaller energy barrier 4.2 Properties and actions of enzymes Not possible because high temperature kill the body cells! By increasing the temperature? smaller energy barrier energy level raised energy barrier is easier to overcome

rate of chemical reactions is speeded up by enzymes (酶) 4.2 Properties and actions of enzymes lower energy barrier rate of chemical reactions is speeded up by enzymes (酶)

rate of chemical reactions is speeded up by enzymes (酶) 4.2 Properties and actions of enzymes energy rate of chemical reactions is speeded up by enzymes (酶)

a Grind fresh liver with cold distilled water. 4.2 Properties and actions of enzymes 4.1 Demonstration of the breaking-down action of enzymes 1 Prepare liver extract. a Grind fresh liver with cold distilled water. fresh pig liver cold distilled water

filter paper liver extract 4.1 4.2 Properties and actions of enzymes 4.1 b Filter the ground tissue with filter paper. filter paper liver extract

distilled water liver extract 4.1 4.2 Properties and actions of enzymes 4.1 c Dilute the liver extract by 50% with cold distilled water. distilled water liver extract

4.2 Properties and actions of enzymes 4.1 2 Set up 3 test tubes A to C. Observe for the release of gas and the gas given off with a glowing splint. hydrogen peroxide + liver extract distilled water + liver extract hydrogen peroxide + distilled water A B C

Results and discussion 4.2 Properties and actions of enzymes 4.1 Results and discussion A gas is released from tube A. The gas is oxygen because it re-lights a glowing splint. No oxygen is released from the control set-ups (tubes B and C).

Results and discussion 4.2 Properties and actions of enzymes 4.1 Results and discussion Fresh liver tissues can break down hydrogen peroxide possibly due to the presence of catalase in the liver tissue. Catalase speeds up the breakdown of hydrogen peroxide.

4.2 Properties and actions of enzymes How do enzymes work? each enzyme has an active site of a specific (專一的) shape active site (活性部位) enzyme

4.2 Properties and actions of enzymes How do enzymes work? active site can combine with substrate (受質) molecule(s) substrate shapes fit together! enzyme

4.2 Properties and actions of enzymes How do enzymes work? active site can combine with substrate (受質) molecule(s) enzyme-substrate complex (酶受質複合物) substrate enzyme complex breaks down to give product(s)

How do enzymes work? in catabolic reactions: substrate 4.2 Properties and actions of enzymes How do enzymes work? in catabolic reactions: substrate enzyme- substrate complex enzyme

How do enzymes work? in catabolic reactions: products 4.2 Properties and actions of enzymes How do enzymes work? in catabolic reactions: products enzyme- substrate complex enzyme unchanged and can be reused

How do enzymes work? in anabolic reactions: substrates 4.2 Properties and actions of enzymes How do enzymes work? in anabolic reactions: substrates enzyme- substrate complex enzyme

How do enzymes work? in anabolic reactions: product 4.2 Properties and actions of enzymes How do enzymes work? in anabolic reactions: product enzyme- substrate complex enzyme unchanged and can be reused

Properties of enzymes 1) Biological catalysts 4.2 Properties and actions of enzymes Properties of enzymes 1) Biological catalysts act as catalysts (催化劑) in organisms speed up metabolic reactions

Properties of enzymes 2) Reusable 4.2 Properties and actions of enzymes Properties of enzymes 2) Reusable enzyme returns to its original form after reaction before reaction after reaction same!

large amount of products produced 4.2 Properties and actions of enzymes Properties of enzymes 3) Required in small amount large amount of products produced reusable reusable reusable

Properties of enzymes 4) Proteins 4.2 Properties and actions of enzymes Properties of enzymes 4) Proteins enzymes are denatured (變性) at high temperatures and extreme pH

Properties of enzymes 5) Specific action different shape! 4.2 Properties and actions of enzymes Properties of enzymes 5) Specific action different shape!

Properties of enzymes 5) Specific action 4.2 Properties and actions of enzymes Properties of enzymes 5) Specific action each enzyme combines with a specific substrate each enzyme catalyses only one type of reaction

Properties of enzymes lock-and-key hypothesis (鎖鑰假說) 4.2 Properties and actions of enzymes Properties of enzymes 5) Specific action can be explained by lock-and-key hypothesis (鎖鑰假說)

Lock-and-key hypothesis 4.2 Properties and actions of enzymes Lock-and-key hypothesis specific shape fit only a particular lock

Lock-and-key hypothesis 4.2 Properties and actions of enzymes Lock-and-key hypothesis active sites of specific shape fit only a particular substrate

4.2 Properties and actions of enzymes 1 Enzymes are biological catalysts that chemical reactions in organisms by lowering the of reactions. They are important in regulating metabolism. speed up energy barrier

enzyme-substrate complex 4.2 Properties and actions of enzymes 2 An enzyme works when its combines with substrate(s) to form an . The complex then breaks down to give the product(s). The enzyme is released in its . active site enzyme-substrate complex original form

3 Enzyme actions are specific because their active sites have 4.2 Properties and actions of enzymes 3 Enzyme actions are specific because their active sites have . Enzyme combines only with substrate(s) that fits into its active site. The specificity of enzyme actions can be explained by the . specific shapes lock-and-key hypothesis

4 Five properties of enzymes: i) They are biological . 4.2 Properties and actions of enzymes 4 Five properties of enzymes: i) They are biological . ii) They can be . iii) They are required in amount. iv) They are . v) Their actions are . catalysts reused small proteins specific

reaction rate of an enzymatic reaction 4.3 Factors affecting the rate of enzymatic reactions Enzyme activity reaction rate of an enzymatic reaction the rate at which substrates are used up or broken down the rate of product formation

reaction rate of an enzymatic reaction 4.3 Factors affecting the rate of enzymatic reactions Enzyme activity enzyme reaction rate of an enzymatic reaction the rate at which substrates are used up or broken down the rate of product formation

temperature temperature pH inhibitor 4.3 Factors affecting the rate of enzymatic reactions enzyme temperature temperature pH inhibitor

Temperature 10 20 30 40 50 60 0˚C low kinetic energy enzyme inactive 4.3 Factors affecting the rate of enzymatic reactions Temperature temperature(℃) reaction rate 10 20 30 40 50 60 0˚C low kinetic energy enzyme inactive

Temperature 10 20 30 40 50 60 temperature rises more kinetic energy 4.3 Factors affecting the rate of enzymatic reactions Temperature temperature(℃) reaction rate 10 20 30 40 50 60 temperature rises more kinetic energy

Temperature more kinetic energy molecules vibrate more rapidly 4.3 Factors affecting the rate of enzymatic reactions Temperature more kinetic energy molecules vibrate more rapidly collide more frequently have higher chance to form an enzyme-substrate complex

Temperature 10 20 30 40 50 60 temperature rises more kinetic energy 4.3 Factors affecting the rate of enzymatic reactions Temperature temperature(℃) reaction rate 10 20 30 40 50 60 temperature rises more kinetic energy reaction rate increases

Temperature 10 20 30 40 50 60 maximum rate optimum temperature 4.3 Factors affecting the rate of enzymatic reactions Temperature temperature(℃) reaction rate 10 20 30 40 50 60 maximum rate optimum temperature rate of enzymatic reaction reaches maximum

4.3 Factors affecting the rate of enzymatic reactions Temperature temperature(℃) reaction rate 10 20 30 40 50 60 temperature higher than optimum temperature enzyme denatured reaction rate decreases

4.3 Factors affecting the rate of enzymatic reactions  Enzymes are killed by heating so the rate of enzymatic reaction decreases.

4.3 Factors affecting the rate of enzymatic reactions Enzymes are NOT living things. They cannot be killed. They are denatured (their shapes are changed) at very high temperatures.

Investigation of the effect of temperature on enzyme activity 4.3 Factors affecting the rate of enzymatic reactions Simulation 4.2 Investigation of the effect of temperature on enzyme activity 1 Add amylase solution to test tubes A to F. Add starch solution to test tubes 1 to 6. 2 Leave the different pairs of test tubes in water baths at different temperatures for 10 minutes.

ice starch solution 0˚C 20˚C 40˚C 60˚C 80˚C 100˚C amylase solution 4.2 4.3 Factors affecting the rate of enzymatic reactions 4.2 ice starch solution A 1 0˚C B 2 20˚C C 3 40˚C D 4 60˚C E 5 80˚C F 6 100˚C amylase solution

amylase solution starch solution 4.2 4.3 Factors affecting the rate of enzymatic reactions 4.2 3 Pour the amylase solution into starch solution. Put the tube of mixture back to its beaker. Record the time as zero. A 1 amylase solution starch solution

amylase and starch mixture iodine drops 4.3 Factors affecting the rate of enzymatic reactions 4.2 4 At 2-minute intervals, transfer a drop of each mixture to an iodine drop. Record the time it takes for the blue-black colour to disappear. amylase and starch mixture iodine drops spot plate

Results and discussion 4.3 Factors affecting the rate of enzymatic reactions 4.2 Results and discussion The time it takes for the blue-black colour to disappear is the shortest at 60˚C. The starch does not disappear at 0˚C, 80˚C and 100˚C.

Results and discussion 4.3 Factors affecting the rate of enzymatic reactions 4.2 Results and discussion At low temperature, the enzymatic reaction rate is low because amylase is inactive. Its activity increases with temperature until it reaches a maximum (around 60˚C).

Results and discussion 4.3 Factors affecting the rate of enzymatic reactions 4.2 Results and discussion The enzyme activity is the highest at 60˚C. Above the optimum temperature, the enzyme activity decreases and the reaction rate decreases until the enzyme becomes denatured and can no longer work.

temperature pH pH inhibitor 4.3 Factors affecting the rate of enzymatic reactions enzyme temperature pH pH inhibitor

many enzymes in mammals (e.g. salivary amylase) reaction rate 4.3 Factors affecting the rate of enzymatic reactions pH many enzymes in mammals (e.g. salivary amylase) pepsin pancreatic lipase 2 4 6 8 10 12 14 pH

pH reaction rate each enzyme works in a narrow range of pH 2 4 6 8 10 4.3 Factors affecting the rate of enzymatic reactions pH each enzyme works in a narrow range of pH 2 4 6 8 10 12 14 pH

pH reaction rate optimum pH for most enzymes: pH 5 – pH 9 4.3 Factors affecting the rate of enzymatic reactions pH optimum pH for most enzymes: pH 5 – pH 9 each enzyme have their own optimum pH unsuitable pH causes denaturation 2 4 6 8 10 12 14 pH

4.3 Factors affecting the rate of enzymatic reactions Simulation 4.3 Investigation of the effect of pH on enzyme activity 1 Add Benedict’s solution to test tubes 1 to 6. Benedict’s solution

4.3 Factors affecting the rate of enzymatic reactions 4.3 2 Add sucrose solution to another 6 test tubes A to F. Then add citrate-phosphate buffer solution at different pH values to the tubes as shown. Tube A B C D E F pH of buffer solution 3.2 4.0 5.2 6.0 7.0 8.0

sucrose solution + citrate-phosphate buffer + invertase 4.3 Factors affecting the rate of enzymatic reactions 4.3 3 Add invertase solution to test tubes A to F. Leave at room temperature for 5 minutes. sucrose solution + citrate-phosphate buffer + invertase

4.3 Factors affecting the rate of enzymatic reactions 4.3 4 Pour the Benedict’s solution in test tubes 1 to 6 into test tubes A to F respectively. Shake the tubes gently. Put the test tubes into a boiling water bath for 10 minutes. boiling water

4.3 Factors affecting the rate of enzymatic reactions 4.3 5 Allow the tubes to stand for 15 minutes. Measure the depth of any brick-red precipitate settled in the test tubes.

Results and discussion 4.3 Factors affecting the rate of enzymatic reactions 4.3 Results and discussion Precipitate is formed in tubes A, B, C and D. The largest amount of precipitate is settled in tube C. No precipitate is formed in tubes E and F. The results show that invertase works in an acidic medium. It has an optimum pH value around pH 5.2.

temperature pH inhibitor inhibitor 4.3 Factors affecting the rate of enzymatic reactions enzyme temperature pH inhibitor inhibitor

4.3 Factors affecting the rate of enzymatic reactions Inhibitors inhibitors (抑制劑) are chemicals that slow down or stop the activities of enzymes

Inhibitors Competitive inhibitors Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions Inhibitors Competitive inhibitors (競爭性抑制劑) Non-competitive inhibitors (非競爭性抑制劑)

1 Competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions Animation 1 Competitive inhibitors competitive inhibitor substrate similar shape!

1 Competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 1 Competitive inhibitors compete with substrates for active sites substrate inhibitor active site enzyme

1 Competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 1 Competitive inhibitors reaction rate decreases inhibitor prevents binding of substrate chance to form enzyme-substrate complex lowered enzyme

1 Competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 1 Competitive inhibitors reversible binding substrate can bind when inhibitor leaves enzyme

1 Competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 1 Competitive inhibitors more substrates, greater chance of binding reaction rate increases

Inhibitors Competitive inhibitors Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions Inhibitors Competitive inhibitors (競爭性抑制劑) Non-competitive inhibitors (非競爭性抑制劑)

2 Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 2 Non-competitive inhibitors non-competitive inhibitor substrate shape not similar do not compete for active sites

2 Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 2 Non-competitive inhibitors active site changes shape

2 Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 2 Non-competitive inhibitors not fit together! reaction rate decreases

2 Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 2 Non-competitive inhibitors increasing substrate concentration cannot increase reaction rate

2 Non-competitive inhibitors 4.3 Factors affecting the rate of enzymatic reactions 2 Non-competitive inhibitors examples: cyanide heavy metals

Investigation of the effect of inhibitors on enzyme activity 4.3 Factors affecting the rate of enzymatic reactions Video 4.4 Investigation of the effect of inhibitors on enzyme activity 1 Add Benedict’s solution to test tubes 1 to 3. 2 Prepare test tubes A to C. A copper(II) suphate +sucrose B silver nitrate + sucrose C distilled water + sucrose

4.3 Factors affecting the rate of enzymatic reactions 4.4 3 Add 10 drops of invertase solution to test tubes A to C. Leave the tubes at room temperature for 5 minutes.

4.3 Factors affecting the rate of enzymatic reactions 4.4 4 Pour Benedict’s solution in test tubes 1 to 3 into test tubes A to C respectively. Shake the tubes gently. Put the test tubes into a boiling water bath for 10 minutes. boiling water

4.3 Factors affecting the rate of enzymatic reactions 4.4 5 Allow the tubes stand for 15 minutes. Measure the depth of any brick-red precipitate settled in the test tubes.

Results and discussion 4.3 Factors affecting the rate of enzymatic reactions 4.4 Results and discussion Precipitate is formed in control set-up (tube C). No precipitate is formed in tubes A and B. The results show that copper(II) ion and silver ion are inhibitors of enzyme invertase. Their presence slows down the action of invertase on sucrose.

1 Effect of temperature on enzymes: Temperature Enzymes 4.3 Factors affecting the rate of enzymatic reactions 1 Effect of temperature on enzymes: Temperature Enzymes Low temperature Work best High temperature Inactive Optimum temperature Denatured

2 Effect of pH on enzymes: pH Enzymes Work best Extreme pH Optimum pH 4.3 Factors affecting the rate of enzymatic reactions 2 Effect of pH on enzymes: pH Enzymes Work best Extreme pH Optimum pH Denatured

Are they similar to substrate? 4.3 Factors affecting the rate of enzymatic reactions 3 Difference between competitive and non-competitive inhibitors: Competitive Non-competitive Shape of molecule Similar Not similar Are they similar to substrate?

3 Non-competitive Competitive 4.3 Factors affecting the rate of enzymatic reactions 3 Difference between competitive and non-competitive inhibitors: Competitive Non-competitive Action Compete for active site Change the shape of enzyme What is their mode of action?

Will the rate of enzymatic reaction increase? 4.3 Factors affecting the rate of enzymatic reactions 3 Difference between competitive and non-competitive inhibitors: Competitive Non-competitive Increased substrate concentration Yes No Will the rate of enzymatic reaction increase?

used to produce useful commercial products 4.4 Applications of enzymes used to produce useful commercial products

biological washing powder 4.4 Applications of enzymes example: lipases and proteases to remove stains containing lipids and proteins biological washing powder

a protease extracted from papaya 4.4 Applications of enzymes example: a protease extracted from papaya papain to soften meat meat tenderizer

extracted from young cows’ stomach 4.4 Applications of enzymes example: extracted from young cows’ stomach proteases to coagulate (凝固) milk in cheese production cheese

to modify starch to keep the bread soft 4.4 Applications of enzymes example: enzymes to modify starch to keep the bread soft bread

to break down plant cell walls so that the juice looks less cloudy 4.4 Applications of enzymes example: enzymes to break down plant cell walls so that the juice looks less cloudy fruit juice

to remove hairs from hides (牛皮) to soften leather 4.4 Applications of enzymes example: enzymes to remove hairs from hides (牛皮) to soften leather leather

What are the advantages of using enzymes? 4.4 Applications of enzymes What are the advantages of using enzymes?

1 Specific in action reduce the production of unwanted products 4.4 Applications of enzymes Advantages Specific in action 1 reduce the production of unwanted products

Efficient in small amount 4.4 Applications of enzymes Advantages Efficient in small amount 2 lower the cost of production

Work at moderate conditions 4.4 Applications of enzymes Advantages Work at moderate conditions 3 extreme and expensive conditions not required e.g. high temperature high pressure

Investigation of protease activities in different fruit juices 4.4 Applications of enzymes 4.5 Investigation of protease activities in different fruit juices 1 Heat the end of a cork borer in a Bunsen flame and allow it to cool.

cork borer well milk agar plate 4.5 4.4 Applications of enzymes 4.5 2 Gently press the borer down into the milk-agar plate to make five wells. Replace the lid quickly. cork borer well milk agar plate

different fruit juices 4.4 Applications of enzymes 4.5 3 Use a clean dropper to fill the wells A–D with pineapple juice, kiwi fruit juice, papaya juice and guava juice. Fill well E with distilled water. different fruit juices distilled water

4.4 Applications of enzymes 4.5 4 Replace the lid. Incubate the plate at 35˚C for one hour. incubator

4.4 Applications of enzymes 4.5 5 Measure the diameter of the clear zones around the wells by placing the plate on graph paper and examining against light.

Results and discussion 4.4 Applications of enzymes 4.5 Results and discussion Clear zones are observed around the wells containing fruit juices and their diameters are different. The well containing distilled water is a control. No clear zone is formed around it.

Results and discussion 4.4 Applications of enzymes 4.5 Results and discussion The results show that pineapple, kiwi fruit, papaya and guava contain proteases that can break down proteins, but the activities of the proteases differ from one another.

4.4 Applications of enzymes Video 4.6 Design an investigation of the effectiveness of different biological washing powder Which brand of biological washing powder is the most effective in removing food stains? Design an investigation to find this out.

washing powder tenderizers 4.4 Applications of enzymes Enzyme are widely used in industry to produce useful products e.g. biological and meat . They are also important in food industries, breweries and leather industries. washing powder tenderizers

1 What are enzymes? enzyme Enzymes are biological catalysts. They speed up metabolic reactions in organisms.

2 What is the importance of enzymes to organisms? Without enzymes, metabolic reactions will not proceed under the relatively low temperatures.

3 What is the advantage of enzyme using enzymes in the making of stonewashed jeans? This can shorten the time of jean production.

Enzymes energy barrier metabolic reactions anabolic reactions lower the energy barrier therefore speed up metabolic reactions include anabolic reactions catabolic reactions

lock-and-key hypothesis Enzymes have an active site shows specificity can be explained by lock-and-key hypothesis

inactivation of enzymes denaturation of enzymes activities affected by temperature pH inhibitors too low causes too high causes extreme values cause inactivation of enzymes denaturation of enzymes

inhibitors competitive non-competitive may be competitive non-competitive