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LECTURE 2:INTRODUCTION TO ENZYME

Space-filling view ofRubisCO showingthe arrangement ofthe large chaindimers (white/grey)and the small chains(blue and orange).from the Protein DataBank

2. Enzymes are the catalysts of biochemical reactions andresponsible for bringing about almost all of the chemicalreactions in living organisms.

3. The life of organisms would be much different and go on veryslow without enzymes

1. The living cell is thesite of tremendousbiochemical activity(biochemicalreactions) which, inmajority, do not takeplace spontaneously,but require catalyststo occur at normalrates.

LECTURE OUTCOMES

After mastering the present lecture materials,students will be able to1. make an enzyme cleaner for use to remove

stains2. explain enzymes as metabolic catalysts to

support certain reactions3. describe the initial discovery of enzymes and

the first crystallization of enzyme4. explain enzyme properties that include enzyme

structure and characteristics

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LECTURE FLOWCompetencyProjectI. INTRODUCTION

1. Definition2. Discovery of

Enzymes2.1 Zymase2.2 Urease2.3 Papain

II. ENZYME PROPERTIESA. Enzyme Structure

1. Proteins2. Cofactors3. Active Site

B. Enzyme Characteristicsa. Critical cell componentsb. Very efficientc. Reduce Gd. Specifice. Subject to regulatory control

PROJECTEnzyme Cleaner

Try Enzyme cleaner to remove stainsThere are many different types of enzymes used incleaning solutions, but the primary enzymes proprietaryblend most use are Protease, Amylase, and Lipase.

Materials1. 300 grams of citrus scraps2. 100 grams of brown sugar3. 1 liter of water4. 2-liter or larger plastic bottle

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Procedure1. 300 g potongan jeruk ditempatkan dalam wadah (botol)2. Tambahkan 100 g gula coklat (brown sugar) ke dalam

botol3. Tuangkan 1 liter air bersih ke dalam botol kemudian tutup4. Kocok campuran bahan hingga gula terlarut dan

tercampur dengan rata yang memakan waktu sekitar 30-60 detik

5. Buat keterangan waktu (hari & bulan) pembuatan bahanpada wadah

6. Simpan wadah pada tempat aman selama 3 bulan untukmendapatkan hasil fermentasi yang baik

7. Bahan kemudian sudah dapat digunakan (300 ml bahan +1 liter air) dengan kualitas yang sama dengan yang dijual

I. INTRODUCTION

1. Definition● Enzymes are metabolic catalysts which

increase the rate of chemical reactions bylowering the activation energy of thatreactions

● An enzymes (in yeast) is made up of proteinthat acts as a catalyst to cause certaindesired reaction

● Enzymes are the largest and most highlyspecialized catalysts in the body for thereactions involved in metabolism

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2. Discovery of enzymes2.1 ZymaseEduard Buchner- Buchner discovered in 1897 that yeast extracts

could ferment sugar to alcohol:C6H12O6 → 2C2H5OH + 2CO2 + 2 ATP

- The conversion of sugar to alcohol (ethanol) iscatalyzed by several enzymes, called zymase, thatare produced by

- Buchner’s finding showed that fermentation waspromoted by molecules that continued to functionwhen removed from cells.

2.2 Urease- In 1926, Sumner isolated and crystallized

jack bean (Canavalia ensiformis) urease,which catalyzes the reaction:

CH4N2O + H2O → 2NH3 + CO2

Kacang parang (Jack bean, Canavalia ensiformis)

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http://www.rcsb.org/pdb/images/3LA4_bio_r_500.jpg?bioNum=1

Crystal structure of the first plant urease from Jack bean(Canavalia ensiformis). DOI: 10.2210/pdb3la4/pdbClassification: HYDROLASEDeposited: 2010-01-06 Released: 2010-07-28Deposition author(s): Ponnuraj, K.Organism: Canavalia ensiformis

Urease active site

Panpae et al., 2012

Enzymes are complexmolecules produced inliving organisms tocatalyze (speed up)chemical reactionswithin the cell

- The urease crystals contained only protein, leadingSumner to propose that all enzymes are proteins.

Crystal structure of apapain-E-64 complex

2.3 PapainWhat is papain? Papain is a protein-cleaving enzyme derived from

papaya and certain other plants.

Varughese et al. (1989)

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Why is meat tough? Muscles have to endure a lot of mechanical stress, and

are made of strong fibers that are hard to cut, and toughconnective tissue holds them together

Individual muscle cells contain microscopic fibrils thatgive them their structural integrity and allow them tocontract.

The fibrils have a complex internal structure boundtogether by long protein chains. The connective tissuethat holds the muscle together is also mostly protein.

How does papain tenderize meat? Papain cuts the protein chains in the fibrils and also in

the connective tissue, disrupting the structural integrityof the muscle fiber, and tenderizing the meat.

3. Enzyme Nomenclature Most enzymes are named for their substrates and

for the reactions that they catalyze, with the suffix"ase" added. (ATPase )

Six major classes of enzymes (Superfamilies:EC 1.1.3.4)

1. Transferases Transfer functional

groups betweenmolecules

2. Oxidoreductases Transfer electrons

(RedOx reactions)3. Hydrolases Break bonds by

adding H2O

4. Lyases Elimination reactions

to form double bonds5. Isomerases Intramolecular

rearangements6. Ligases Join molecules with

new bonds

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TransferasesAminotransferases: transferases catalyzing the amino acid degradationby removing amino groups.

OxidoreductasesAlcohol dehydrogenase: an oxidoreductase converting alcohols toaldehydes/ ketones.

HydrolasesGlucose-6-phosphatase: a hydrolase that removes the phosphate groupfrom glucose-6-phosphate, leaving glucose and H3PO4.

LyasesPyruvate decarboxylase: a lyase that removes CO2 from pyruvate.

IsomerasesRibulose phosphate epimerase: an isomerase that catalyzes theinterconversion of ribulose-5-phosphate and xylulose-5-phosphate.

LigasesHexokinase: a ligase that catalyzes the interconversion of glucose andATP with glucose-6-phosphate and ADP.

2. ENZYME PROPERTIESA. Enzyme Structure

1. Proteins2. Cofactors3. Active Site

B. Enzyme Characteristicsa. Critical components of cells

Almost all reactions in living organism/plants are catalyzedby enzymes)

b. Very efficientc. Reduce G for reaction (by binding the transition

state)d. Specifice. Subject to regulatory control of various sorts

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A. Enzyme StructureEnzyme consists of protein with active siteand cofactor (coenzyme)

NADH

Apoenzyme + Coenzyme = Holoenzyme

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1. Protein All known enzymes are high molecular weight

compounds that are made up principally of proteins(except for catalytic RNA)

Apoenzyme: The polypeptide orprotein part of the enzyme whichmay be inactive in its originalsynthesized structure

The inactive form of theapoenzyme is known as aproenzyme or zymogen.

The proenzyme may contain several extra aminoacids in the protein which are removed, and allowsthe final specific tertiary structure to be formed beforeit is activated as an apoenzyme.

2. Cofactors Many enzymes require the presence of other

compounds - cofactors - before their catalytic activitycan be exerted

This entire active complex is referred to as theholoenzyme; apoenzyme (protein portion) + thecofactor (coenzyme, prosthetic group or metal-ion-activator)

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Apoenzyme + Cofactor = HoloenzymeThe cofactor may be:1. A coenzyme - a non-protein organic substance which is

loosely bound to the protein part, dialyzable, andthermostable

2. A prosthetic group - an organic substance which isfirmly bound to the protein or apoenzyme portion,dialyzable and thermostable.

3. A metal-ion-activator - these include K+, Fe++, Fe+++,Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++

3. Enzyme Active Sites● The active site is the specific area of the enzyme to which the

substrate attaches during the reaction part of the conformation of the enzyme molecule

arranged to create a special pocket or cleftwhose three-dimensional structure iscomplementary to the structure of the substrate

● The enzyme and the substrate molecules"recognize" each other through this structuralcomplementarity

● The substrate binds to the enzyme through relativelyweak forces -H bonds, ionic bonds (salt bridges),and van der Waals interactions between stericallycomplementary clusters of atoms.

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Lysozyme active site: Green showssubstrate contacts and orange arecatalytic residues

Sucraseactive site

B. Characteristicsa. Critical Components

Enzymes are critical forevery aspect of cellular life(cell metabolism &biological processes)

b. Very efficient catalystsA very small quantity ofan enzyme can catalyzethe transformation ofvastly lager quantity ofthe substrate Sucrase Catalase

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Sucrase (invertase) can effect the hydrolysis ofat least 1,000,000 times its own weight ofsucrose without exhibiting any appreciablediminution in its activity

Catalase is one of the more efficient enzymes,one molecule of this enzyme being able tocatalyze the conversion 5,000,000 molecules ofH202 per minute (the reduction of hydrogenperoxide to water and molecular oxygen) whenconditions are favorable

Enzyme Reactioncatalyzed

Function ofreaction

Rate*

Catalase 2H2O2 2H2O + O2removes toxic H2O2from cell 1015

Carbonicanhydrase CO2 + H2O H2CO3

hydrates CO2 gasfor transport 107

* rate of enzyme catalyzed compared to rate uncatalyzed

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c. Activation Energy Enzymes reduce the activation energy of a

reaction so that the kinetic energy of mostmolecules exceeds the activation energyrequired and so they can react. For example, for the catalase reaction

2H2O2 → 2H2O + O2The activation energy is86 kJ mol-1 with no catalyst62 kJ mol-1 with an inorganic catalyst1 kJ mol-1 with the enzyme catalase

RATE of reaction is affected by enzyme RATE depends on ΔG‡, the Arrhenius activation

energy (i.e., the free energy of activation for thereaction).

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Gibb’s Free Energy Change (G) Enzymes decrease activation energy

(ΔG‡) for reactions catalyzed. K’°eq = [P]/[S] From thermodynamics, the

equilibrium constant and the freeenergy are related by the expressionΔG’° = -RTln K’°eq

= -2.3RTlog K’°eq ΔG = overall difference in free energy

between final (P) and starting (S), notaffected by enzyme.

K’eq

∆G'0(KJ/mol)

10-6 34.210-5 28.510-4 22.810-3 17.110-2 11.410-1 5.7

1 010 -5.7102 -11.4103 -17.1

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Enzymatic catalysts have much higher ratesthan non-enzymatic catalysts do, and even atrelatively low temperatures

UREA FERTILIZER The optimum conditions for enzyme catalysis are almost

invariably moderate temperatures, and pHs which are notextreme

The contrast between a reaction catalysed by an enzymeand by a non-enzymatic catalyst is well illustrated by theprocess of nitrogen fixation (i.e. reduction of N2 toammonia). Nitrogenase catalyses this reaction attemperatures around 300 K and at neutral pH. The enzymeis a complex system comprising two dissociating proteincomponents one of which contains iron and the other ironand molybdenum. Several molecules of ATP arehydrolyzed during the reduction

By contrast, in the industrial synthesis of ammonia fromnitrogen and hydrogen, the conditions used are as follows:temperature 700 - 900 K, pressure 100 - 900 atmospheres,and the presence of an iron catalyst, often promoted bytraces of oxides of other metals

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d. Enzyme SpecificityEnzymes very specific– for substrate acted upon– for reaction catalyzedExample: Proteases are a whole class of enzymes that

all catalyze hydrolysis of peptide bonds:

Specificity class● Absolute specificity - the enzyme will catalyze only

one reaction.● Group specificity - the enzyme will act only on

molecules that have specific functional groups, suchas amino, phosphate and methyl groups.

● Linkage specificity - the enzyme will act on aparticular type of chemical bond regardless of therest of the molecular structure.

● Stereochemicalspecificity - the enzymewill act on a particularsteric or optical isomer.

Enzyme Specificity

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e. Enzyme RegulationEnzymes are tightly regulated light switchesUnregulated enzymes become constitutively

active or inactive (light is always on or off )Unregulated enzyme activity disrupts cell

homeostasis and often lead to disease states.Unregulated enzyme

activity disrupts cellhomeostasis andoften lead to diseasestates.