LECTURE 2: : : : INTRODUCTION TO INTRODUCTION TO INTRODUCTION TO INTRODUCTION TO

ENZYMEENZYMEENZYMEENZYME

Ribonuclease structure showing histidine probes

Enzymes are critical for every aspect of cellular life

What is an important enzyme that you are

familiar with?

PAPAINPAPAINPAPAINPAPAIN

• What is papain?What is papain?What is papain?What is papain?

– Papain is a protein-cleaving

enzyme derived from papaya enzyme derived from papaya

and certain other plants.

Enzymes are complex

molecules produced in living

organisms to catalyze (speed

up) chemical reactions within

the cell

PAPAINPAPAINPAPAINPAPAIN

• Why is meat tough?Why is meat tough?Why is meat tough?Why is meat tough?

– Muscles have to endure a lot of mechanical stress; they are

made of strong fibers that make them hard to cut, and

tough connective tissue holds them together. Individual

muscle cells contain microscopic fibrilsfibrilsfibrilsfibrils that give them their

structural integrity and allow them to contract. The fibrils

have a complex internal structure bound together by long have a complex internal structure bound together by long

protein chains. The connective tissue that holds the muscle

together is also mostly protein.

• How does papain tenderize meat?How does papain tenderize meat?How does papain tenderize meat?How does papain tenderize meat?

– Papain cuts the protein chains in the fibrils and also in the

connective tissue, disrupting the structural integrity of the

muscle fiber, and tenderizing the meat.

ENZYMES ARE METABLOLIC CATALYSTS.

1. DEFINITION

Enzymes (in yeast) are the largest and most highly specialized catalysts in the body for the reactions involved in metabolism whichincrease the rate of chemical reactions by increase the rate of chemical reactions by lowering the activation energy of that reactions

ENZYME TOPICSENZYME TOPICSENZYME TOPICSENZYME TOPICS

1. Functional Properties

2. Nomenclature

3. Enzyme Specificity3. Enzyme Specificity

4. Enzyme Regulation

5. Activation Energy

1. Enzyme PropertiesENZYMES ARE1. Proteins (note that recent developments indicate that both

RNA and antibodies may have catalytic activity, these are

called ribozymes, and catalytic antibodies or abzymes,

respectively)

2. Biological catalysts, critical components of cell metabolism &

biological processes.

3. Very efficient catalysts

4. Reduce ∆G for reaction (by binding the transition state)

5. Subject to regulatory control of various sorts

6. Carry out catalysis in a special region of the molecule, the

active-site

7. Exhibit special kinetics

Very efficient catalysts A very small quantity of an enzyme can catalyze the A very small quantity of an enzyme can catalyze the A very small quantity of an enzyme can catalyze the A very small quantity of an enzyme can catalyze the transformation of vastly lager quantity of the substratetransformation of vastly lager quantity of the substratetransformation of vastly lager quantity of the substratetransformation of vastly lager quantity of the substrate

� SucraseSucraseSucraseSucrase ((((invertaseinvertaseinvertaseinvertase) ) ) ) can effect the hydrolysis of at least

1,000,000 times its own weight of sucrose without

exhibiting any appreciable diminution in its activity

� CatalaseCatalaseCatalaseCatalase is one of the more efficient enzymes, one

molecule of this enzyme being able to catalyze the molecule of this enzyme being able to catalyze the

conversion 5,000,000 molecules of H202 per minute (the

reduction of hydrogen peroxide to water and molecular

oxygen) when conditions are favorable

THE HYDROLISIS OF SUCROSE CATALYZED BY SUCRASE

� Kecepatan reaksi sederhana hidrasi karbon dioksida meningkat 107 kali lipat dengan enzimCarbonic anhydrase dibadingkan dengan tanpa

enzim

CO2 + H2O H2CO3Setiap molekul Carbonic anhydrase dapat

menghidrasi 105 molekul CO2 per detik2

� Kecepatan reaksi dengan

� hexokinase >>>>1010

� phosphorylase >>>>3.1011

� alcohol dehydrogenase >>>>2.108

� creatine kinase >>>>104.

Enzymatic catalysts have much higher rates than nonEnzymatic catalysts have much higher rates than nonEnzymatic catalysts have much higher rates than nonEnzymatic catalysts have much higher rates than non----enzymatic enzymatic enzymatic enzymatic

catalysts do, and even at relatively low temperatures (Table 1)catalysts do, and even at relatively low temperatures (Table 1)catalysts do, and even at relatively low temperatures (Table 1)catalysts do, and even at relatively low temperatures (Table 1)

UREA FERTILIZERUREA FERTILIZERUREA FERTILIZERUREA FERTILIZER• The optimum conditions for enzyme catalysis are almost invariably The optimum conditions for enzyme catalysis are almost invariably The optimum conditions for enzyme catalysis are almost invariably The optimum conditions for enzyme catalysis are almost invariably

moderate temperaturesmoderate temperaturesmoderate temperaturesmoderate temperatures, and , and , and , and pHs which are not extremepHs which are not extremepHs which are not extremepHs which are not extreme

• The contrast between a reaction catalysed by an enzyme and by a nonThe contrast between a reaction catalysed by an enzyme and by a nonThe contrast between a reaction catalysed by an enzyme and by a nonThe contrast between a reaction catalysed by an enzyme and by a non----

enzymatic catalyst is well illustrated by the process of nitrogen fixation enzymatic catalyst is well illustrated by the process of nitrogen fixation enzymatic catalyst is well illustrated by the process of nitrogen fixation enzymatic catalyst is well illustrated by the process of nitrogen fixation

(i.e. reduction of N2 to ammonia). Nitrogenase catalyses this reaction at (i.e. reduction of N2 to ammonia). Nitrogenase catalyses this reaction at (i.e. reduction of N2 to ammonia). Nitrogenase catalyses this reaction at (i.e. reduction of N2 to ammonia). Nitrogenase catalyses this reaction at

temperatures around temperatures around temperatures around temperatures around 300 K300 K300 K300 K and at and at and at and at neutral pHneutral pHneutral pHneutral pH. The enzyme is a complex . The enzyme is a complex . The enzyme is a complex . The enzyme is a complex

system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which system comprising two dissociating protein components one of which

contains iron and the other iron and molybdenum. Several molecules of contains iron and the other iron and molybdenum. Several molecules of contains iron and the other iron and molybdenum. Several molecules of contains iron and the other iron and molybdenum. Several molecules of

ATP are hydrolyzed during the reductionATP are hydrolyzed during the reductionATP are hydrolyzed during the reductionATP are hydrolyzed during the reduction

• By contrast, in the industrial synthesis of ammonia from nitrogen and By contrast, in the industrial synthesis of ammonia from nitrogen and By contrast, in the industrial synthesis of ammonia from nitrogen and By contrast, in the industrial synthesis of ammonia from nitrogen and

hydrogen, the conditions used are as follows: temperaturehydrogen, the conditions used are as follows: temperaturehydrogen, the conditions used are as follows: temperaturehydrogen, the conditions used are as follows: temperature 700 700 700 700 ---- 900 K900 K900 K900 K, , , ,

pressure pressure pressure pressure 100 100 100 100 ---- 900900900900 atmospheresatmospheresatmospheresatmospheres, and the presence of an , and the presence of an , and the presence of an , and the presence of an iron catalystiron catalystiron catalystiron catalyst, often , often , often , often

promoted by promoted by promoted by promoted by traces of oxides of other metalstraces of oxides of other metalstraces of oxides of other metalstraces of oxides of other metals

2. Enzyme Nomenclature

Superfamilies: EC 1.1.3.4• Transferases

– Transfer functional groups between molecules• Oxidoreductases

– Transfer electrons (RedOx reactions)• Hydrolases

– Break bonds by adding H2O– Break bonds by adding H2O• Lyases

– Elimination reactions to form double bonds• Isomerases

– Intramolecular rearangements• Ligases

– Join molecules with new bonds

Enzyme Nomenclature

3. Enzyme Specificity

The active site of an enzyme dictates specificity• Tend to be specific for one type of chemical group

• Substrates have to interact in stereospecific manner (fit)

• Substrates have to bind relatively well (affinity)– H-bonds, electrostatics, hydrophobicity

• Substrates have to react– bonds to be broken or formed have to have proper reactivity

– Substances that fit and bind but don’t react are inhibitors

Stereospecificity of Enzymes

1.1.1.1. Each enzyme catalyzes only one type of reaction, and Each enzyme catalyzes only one type of reaction, and Each enzyme catalyzes only one type of reaction, and Each enzyme catalyzes only one type of reaction, and

in some cases will limit its activity to only one in some cases will limit its activity to only one in some cases will limit its activity to only one in some cases will limit its activity to only one

particular type of reactant moleculeparticular type of reactant moleculeparticular type of reactant moleculeparticular type of reactant molecule

Incomparison, platinum catalyzes several different Incomparison, platinum catalyzes several different Incomparison, platinum catalyzes several different Incomparison, platinum catalyzes several different

The specificity of enzymes for reactions is high different from The specificity of enzymes for reactions is high different from The specificity of enzymes for reactions is high different from The specificity of enzymes for reactions is high different from

inorganic catalysts.inorganic catalysts.inorganic catalysts.inorganic catalysts.

types of reactions. types of reactions. types of reactions. types of reactions.

Enzim proteolitik, sebagai contoh, mengkatalisis hidrolisis ikatan peptida. Kebanyakan enzim proteolitik juga mengkatalisis reaksi yang berbeda tapi berhubungan yaitu hidrolisis ikatan ester

Gambar 1. Hidrolisis ikatan peptida dan ester

2. Different end products are formed from the same substrate under the influence of different enzymes.� The trisaccharide raffinose is hydrolizedinto melibiose and fructose in the presence of sucrase, while, the end products of the reaction are sucrose and products of the reaction are sucrose and galactose in the presence of emulsin

� Pyruvic acid is notable for the large number of compounds into which it may be converted by the action of different enzymes

3. Sebagian enzim disintesis dalam bentuk yang tidak aktif, dan dikatifkan kemudian pada waktu dan tempat yang tepat secara fisiologi

– This regulation is effected through changes in the catalytic activity of early enzymes in the pathway, carbamoyl-phosphate synthetase pathway, carbamoyl-phosphate synthetase and aspartate carbamoyltransferase

– Enzim pencernaan seperti trypsinogendisintesis dalam pankreas dan diaktifkan oleh pemotongan ikatan peptida dalam usus (intestine) kecil untuk membentuk enzim aktif trypsin

4. The phenomenon of feedback inhibition is common in many biosynthetic pathways.

– In the biosynthetic pathway leading to the synthesis of pyrimidine nucleotidcs, the end products UTP and CTP are able to end products UTP and CTP are able to inhibit the first enzyme in the pathway; thus they are able to limit the flow of metabolites into that pathway and so regulate their own biosynthesis

Enzyme Active SitesEnzyme Active SitesEnzyme Active SitesEnzyme Active Sites

− The active site is the specific area of the enzyme to The active site is the specific area of the enzyme to The active site is the specific area of the enzyme to The active site is the specific area of the enzyme to which the substrate attaches during the reactionwhich the substrate attaches during the reactionwhich the substrate attaches during the reactionwhich the substrate attaches during the reaction

− The active site is part of the conformation of the The active site is part of the conformation of the The active site is part of the conformation of the The active site is part of the conformation of the enzyme molecule arranged to create a special pocket or enzyme molecule arranged to create a special pocket or enzyme molecule arranged to create a special pocket or enzyme molecule arranged to create a special pocket or cleft whose threecleft whose threecleft whose threecleft whose three----dimensional structure is dimensional structure is dimensional structure is dimensional structure is complementary to the structure of the substratecomplementary to the structure of the substratecomplementary to the structure of the substratecomplementary to the structure of the substrate

− The enzyme and the substrate molecules "recognize" The enzyme and the substrate molecules "recognize" The enzyme and the substrate molecules "recognize" The enzyme and the substrate molecules "recognize" − The enzyme and the substrate molecules "recognize" The enzyme and the substrate molecules "recognize" The enzyme and the substrate molecules "recognize" The enzyme and the substrate molecules "recognize" each other through this structural complementarityeach other through this structural complementarityeach other through this structural complementarityeach other through this structural complementarity

− The substrate binds to the enzyme through relatively The substrate binds to the enzyme through relatively The substrate binds to the enzyme through relatively The substrate binds to the enzyme through relatively weak forces weak forces weak forces weak forces ----H bonds, ionic bonds (salt bridges), and H bonds, ionic bonds (salt bridges), and H bonds, ionic bonds (salt bridges), and H bonds, ionic bonds (salt bridges), and van der Waals interactions between sterically van der Waals interactions between sterically van der Waals interactions between sterically van der Waals interactions between sterically complementary clusters of atoms.complementary clusters of atoms.complementary clusters of atoms.complementary clusters of atoms.

Lysozyme active site: Lysozyme active site: Lysozyme active site: Lysozyme active site: Green shows Green shows Green shows Green shows substrate contacts and orange are substrate contacts and orange are substrate contacts and orange are substrate contacts and orange are catalytic residuescatalytic residuescatalytic residuescatalytic residues

Active site complements structure of Active site complements structure of Active site complements structure of Active site complements structure of substratesubstratesubstratesubstrate

Contain amino acids that function in Contain amino acids that function in Contain amino acids that function in Contain amino acids that function in substrate binding, chemical catalysis, substrate binding, chemical catalysis, substrate binding, chemical catalysis, substrate binding, chemical catalysis, and product releaseand product releaseand product releaseand product release

• X-ray crystallography (also NMR); physical methods to solve structure of enzymes

• Conformation with or without substrate provides functional/ biological information

Enzyme Three Dimensional Structure

biological information

• Used to identify amino acids involved in catalysis

• Example: Prostaglandin Synthase I with arachidonic acid

• PGHS (COX) target of aspirin

Enzyme Three Dimensional Structure

• Enzymes are tightly regulated light switchesEnzymes are tightly regulated light switchesEnzymes are tightly regulated light switchesEnzymes are tightly regulated light switches

• Unregulated enzymes become constitutively Unregulated enzymes become constitutively Unregulated enzymes become constitutively Unregulated enzymes become constitutively

active or inactive (light is always on or off)active or inactive (light is always on or off)active or inactive (light is always on or off)active or inactive (light is always on or off)

• Unregulated enzyme activity disrupts cell Unregulated enzyme activity disrupts cell Unregulated enzyme activity disrupts cell Unregulated enzyme activity disrupts cell

4. Enzyme Regulation4. Enzyme Regulation4. Enzyme Regulation4. Enzyme Regulation

• Unregulated enzyme activity disrupts cell Unregulated enzyme activity disrupts cell Unregulated enzyme activity disrupts cell Unregulated enzyme activity disrupts cell

homeostasis and often lead to disease states.homeostasis and often lead to disease states.homeostasis and often lead to disease states.homeostasis and often lead to disease states.

5. Energi Aktivasi

1. Kecepatan reaksi yang tinggi dengan keberadaan enzim berhubungan dengan energi aktivasi

– Jika reaksi yang terjadi dalam sel berlangsung diluar sel, kecepatannya akan sangat lambat diluar sel, kecepatannya akan sangat lambat kecuali energi diberikan, misalnya dengan peningkatan suhu. Sementara reaksi dalam sel berlangsung pada suhu sekitar lingkungannya (mis. 5o - 40oC).

ACTIVATION ENERGY

2. Kecepatan reaksi kimia yang tinggi pada suhu kamar (mis. laboratorium) tidak mungkin terjadi

� Karena kebanyakan reaksi kimia, sekalipun mengeluarkan energi, tidak terjadi secara mengeluarkan energi, tidak terjadi secara spontan (berlangsung dengan sendirinya) tetapi membutuhkan tambahan energi yang disebut energi aktivasi (energy of activation).

QUIZ

1. Define metabolism and anabolic and catabolic reactions

2. Identify the parts of an enzymes catalyze reactions

3. Describe method by which enzymes catalyze reactions

4. Explain the lock-and-key and induced fit models of enzyme action

5. Explain how changes in pH and temperature will affect enzyme activityenzyme activity

6. Define vitamin, and explain why vitamins are essential for normal cellular function

7. Describe the function of hormones in the living organism

8. Define multienzyme system, and explain how such systems are regulated

9. Describe the ways in which enzyme activity can be inhibited, and give examples of each type of inhibition