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Enzim

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History of enzyme

Energy and life

How enzyme work

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Cells and Energy Chapter 5

~Flow of Energy in Living Things

~The Laws of Thermodyamics

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What is Energy?

Capacity to do work

Forms of energy Potential energy

Kinetic energy

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5.1 The Flow of Energy

in Living Things

Energy

Kinetic and potential

Fig. 5.1

Potential

Energy =storedenergy

Kinetic

Energy =energy of

motion

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5.1 The Flow of Energy

in Living Things

Forms of energy:

Mechanical, sound, light, electric, heat, etc.

Thermodynamics

Study of energy

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5.1 The Flow of Energy

in Living Things

Photosynthetic organisms

SUN

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5.2 The Laws of Thermodynamics

The First Law Energy cannot be created or destroyed

Energy is converted

Heat energy

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Second Law of

Thermodynamics

No energy conversion is ever 100 % efficient

High quality energy is converted to low quality

energy.

Life is a constant battle against the second law.

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The Second Law of Thermodynamics

Disorder increases

Entropy

Fig. 5.3

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Entropy

Measure of degree of disorder in a system

The world of life can resist the flow towardmaximum entropy only because it is

resupplied with energy from the sun

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5.3 Chemical Reactions

Chemical reactions Reactants(substrates)Products

Exergonic reactions

Endergonic reactions

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Energy of Activation

Fig. 5.4

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Catalysts

Fig. 5.4

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Catalysts

speed up the rate of the reaction.

lowers the energy input required for achemical reaction to happen

remains unchanged at the end of thereaction

skool

More free powerpoints at www.worldofteaching.com

http://lgfl.skoool.co.uk/keystage3.aspx?id=63http://lgfl.skoool.co.uk/keystage3.aspx?id=63

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The study history of

enzymes

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The discovery of enzymes as thebiocatalysts (1)

Both enzymology and biochemistry wereevolved from the 19th centuryinvestigation on the nature ofanimaldigestion and fermentation.

Biochemical reactions could not bereproduced in the lab initially and wasthought (e.g., Louis Pasteur) to occur by

the action of a vital force.

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The discovery of enzymes as thebiocatalysts (1)

The idea of catalytic force or contactsubstance promoting fermentation wasintroduced in about 1830s.

Addition of alcohol to an aqueous extractof malt (geminating barley) and salivaprecipitated a flocculent () materialwhich liquefied starch paste and convertedit into sugar, this material was nameddiastase(1833) (lateramylase).

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The discovery of enzymes as thebiocatalysts (2)

Pepsin was discovered as the activeprinciple in the acid extract of gastricmucosa causing the dissolution of

coagulated egg white (1834). Other soluble ferments discovered in the

19th century include trypsin (1857), invertin

(later invertase and sucrase, 1864), papain(vegetable trypsin, 1879), etc.

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The discovery of enzymes as thebiocatalysts (2)

Enzyme (something in yeast) wasfirst coined for such unorganizedferments by Khne in 1876.

Enzymes for alcoholic fermentationwere found to be active in cell freeextracts of yeast (1897, Eduard

Buchner): fermentation is a chemicalprocess, not a vital process.

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The discovery of enzymes as thebiocatalysts (3)

Relationship of initial velocity (V0) andsubstrate concentration (S) wasexamined.

A mathematical description wasestablished for the kinetics of enzymeaction (Michaelis and Menten, 1913).

Th di f th

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The discovery of enzymes as thebiocatalysts (3)

Weak-bonding interactions between the enzymesand their substrates were proposed to distort thesubstrate and catalyze a reaction (Haldane, 1930s).

A GermanA Canadian

A BritishGeneticist

Before it was known that enzymes are proteins!!!

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The Nobel Prize in Chemistry 1907

"for his biochemical

researches and his discoveryof cell-free fermentation"

Eduard Buchner

Germany

Landwirtschaftliche Hochschule

(Agricultural College) Berlin,

Germany

b. 1860, d. 1917.

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Enzyme specificity was revealedby studying sugar conversion

(Emil Fischer, 1890S) Sugars of known structure were synthesized and

used as substrates of enzymes.

The -methylglucoside was found to behydrolyzed by invertin, but not by emulsin,whereas the -methylglucoside was cleaved byemulsin, but not by invertin: the enzyme and the

glucoside was considered to fit (complement)each other like a lock and a key.

Formation of an ES complex was proposed (1894)

E f d b

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Enzymes were found to beproteins

The question of homogeneity of the enzyme preparationsfrustrated the field of enzymology for many decades.

Nitrogen content analysis and various color tests (forproteins) led to contradictory results.

Filterable coenzymes (co-ferments) were discovered inBuchners zymase (Harden and Young, 1906).

Enzymes were thought to be small reactive moleculesadsorbed on inactive colloidal material, including proteins

( as by R. Willsttter in the 1920s). Urease (1926, Sumner) and pepsin (1930, Northrop) were

crystallized and found to be solely made of proteins.

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Urease crystals( X 728)

Sumner, J. B. (1926) Theisolation and

crystallization of theenzyme urease J. Biol.

Chem. 69:435-441.

P i t l

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Pepsin crystals(X90)

Northrop, J. H. (1930)Crystallin pepsin, 1:Isolation and tests ofpurity J. Gen . Physiol.13:739-766.

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The Nobel Prize in Chemistry 1946

for his discovery

that enzymes

can becrystallized"

"for their preparation of enzymes andvirus proteins in a pure form"

James BatchellerSumner John Howard Northrop Wendell MeredithStanley

1/2 of the prize 1/4 of the prize 1/4 of the prize

Cornell UniversityIthaca, NY, USA

Rockefeller Institute forMedical ResearchPrinceton, NJ, USA

Rockefeller Institute forMedical ResearchPrinceton, NJ, USA

1887-1955 1891-1987 1904-1971

Not all enzymes are proteins: Some RNA

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Not all enzymes are proteins: Some RNAmolecules (ribozymes) were found to be

catalytic (Sidney Altman and Thomas Cech,

1981).Ribozymes are found to promote RNA

processing.

Sidney Altman visiting PKU

F ti f b t t

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Formation of an enzyme-substrate(ES) complex was suggested

The activity of invertase in the presence of sucrosesurvives a temperature that completely destroys it if thesucrose is not present (C OSullivan and F. W. Tompson,

1890).

Emil Fishers study on the specifity of invertase (1894).

The rate of fermentation of sucrose in the presence ofyeast seemed to beindependent of the amount of sucrosepresent, but on the amount of the enzyme (A. J. Brown,1902).

The kinetics of enzyme action was originally studied usinginvertase (a hyperbola when V0was plotted against [S]).

The enzyme (E) was thus assumed to form a complex. (ES)with the substrate (S) before the catalysis.

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The kinetics of the enzyme-catalyzed reaction were found to

be rather different from those of atypical chemical reaction

The rate is proportional to the

concentration of the reactantin a typical chemical reaction.

Enzymes however showed

a saturation kinetics:formation of ES complexwas hypothesized (1902).

Th th f lif

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The enzyme theory of life wasformulated

Enzymes are central to every biochemicalprocess (Hofmeister, 1901): life is short andthus has to be catalyzed.

Isolation, purification and physico-chemical

characterization of enzymes would beimportant for understanding the nature of life. Without catalysis, the chemical reactions

needed to sustain life could not occur on a

useful time scale. Self replication and catalysis are believed to be

the two fundamental conditions for life to beevolved. (RNA is thus proposed to be the type

of life molecules first evolved).

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What is enzyme?

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Enzymes

Enzymes are Biological catalysts

Enzymes control chemical reactionsthat take place in the cytoplasm.http://programs.northlandcollege.edu/biology/Biology1111/animations/enz

yme.html

Catalase in an example of an enzymemade by living cells

http://programs.northlandcollege.edu/biology/Biology1111/animations/enzyme.htmlhttp://programs.northlandcollege.edu/biology/Biology1111/animations/enzyme.htmlhttp://programs.northlandcollege.edu/biology/Biology1111/animations/enzyme.htmlhttp://programs.northlandcollege.edu/biology/Biology1111/animations/enzyme.html

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Susunan enzim

Komponen utama enzim adalah protein Protein yang sifatnya fungsional, bukan

protein struktural

Tidak semua protein bertindak sebagaienzim

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Sifat enzim

Enzim dibentuk dalam protoplasma sel

Enzim beraktifitas di dalam sel tempat

sintesisnya (disebut endoenzim) maupun

di tempat yang lain diluar tempat

sintesisnya (disebut eksoenzim)

Sebagian besar enzim bersifat endoenzim

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How enzyme works?

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Substrate Binding

Unlike inorganic catalysts,enzymes are specific

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Unlike inorganic catalysts,enzymes are specific

succinic dehydrogenase

HOOC-HC=CH-COOH HOOC-CH2-CH2-COOH+2H

fumaric acid succinic acid

NOT a substrate for the enzyme:

1-hydroxy-butenoate: HO-CH=CH-COOH

(simple OH instead of one of the carboxyl's)

Maleic acid

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Dlm system biologi reaksi

kimia selalu memerlukan katalis.

Tanpa katalis sangat lama

shg diperlukan Enzim ygberfungsi sbg biokatalisator

protein yang berfungsi untuk

mempercepat reaksi dengan jalan

menurunkan tenaga aktivasi dantidak mengubah kesetimbangan

reaksi, serta bersifat sangat

spesifik.

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Sifat enzim

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Katalis yg paling

efisien mampu

mempercepat reaksi1020 kali lbh cepat

Enzim bersifat sangat

spesifik, baik jenisreaksi maupun

substratnya ,

Tripsin

Trombin

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Enzim tidak ikut bereaksi dgn substrat atauproduknya (struktur enzim tidak berubah baiksebelum dan sesudah reaksi tetap)

Aktifitas dapat dikontrol sesuai dengankebutuhan organisme itu sendiri

Contoh : enzim yg mengkatalisis reaksipertama pada suatu siklus biosintesis biasanya

di hambat oleh produk akhirnya(feedbackinhibition)

Bagian enzim yang aktif adalah sisi aktif darienzim

bbrp enzim disintesis dlm btk tidak aktif. Danakan diaktifkan oleh kondisi dan waktu yangsesuai (enzim allosterik) . prekursor yg tidakaktif disebutzymogen

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Tiga sifat utama enzim :

Kemampuan katalitiknya

Spesifisitas

Kemampuan untuk diatur (regulasi)

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Bagian-bagian enzim

Beberapa stilah:

Holoenzim

Apoenzim/ apoprotein

Gugus prostetik

Koenzim

Kofaktor

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Bagian-bagian enzim (lanjutan)

Seperti halnya protein lain, enzim memiliki BMantara 12,000 1 juta kd

Beberapa enzim tidak membutuhkan molekulkimiawi lain untuk aktifitasnya, beberapamembutuhkan kofaktor / koenzim

Kofaktor ion-ion inorganik yg dibutuhkanenzim untuk melakukan fungsinya

Koenzim

molekul organik (komplek) yangdibutuhkan enziim untuk melakukan fungsinya

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Protein Enzim protein

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Enzim

Protein psederhana

Protein +Bukan Protein

Protein = apoenzim

EnzimKonjugasi

Bukan protein =

Gugus prostetik

Organik =

Koenzim

Anorganik = kofaktor

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f

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Klasifikasi enzim

Klas Tipe reaksi

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Oksidoreduktase

(nitrat reduktase)

memisahkan dan menambahkan elektron atau

hidrogen

Transferase

(Kinase)

memindahkan gugus senyawa kimia

Hidrolase

(protease, lipase,

amilase)

memutuskan ikatan kimia dengan

penambahan air

Liase

(fumarase)

membentuk ikatan rangkap dengan

melepaskan satu gugus kimia

Isomerase

(epimerase)

mengkatalisir perubahan isomer

Ligase/sintetase

(tiokinase)

menggabungkan dua molekul yang disertai

dengan hidrolisis ATP

Polimerasetiokinase menggabungkan monomer-monomersehin a terbentuk olimer

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Reaksi tanpa enzim:

Lambat

Membutuhkan suhu yang tinggi

Tekanan yang tinggi

Reaksi enzimatis

Enzim memberikan suatu lingkungan yg

spesifik di dalam sisi aktifnya, sehingga reaksisecara energetik dapat lebih mudah terjadi

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Perbedaan antara energi reaktan (fase awal)

dgn energi produk (fase akhir) selisih energi

bebas standar (

G)

Agar reaksi berjalan

spontan, bagaimanakah

nilai G

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Enzim mempercepat reaksi tetapi tidak mengubah

keseimbangan reaksi atau G

Kesetimbangan reaksi antara Reaktan dan produk

mencerminkan perbedaan energi bebas pada fase awal

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Kecepatan reaksi tergantung energi aktifasi G suatu pasokan energi dibutuhkan untuk mengawali suatu

reaksi

Energi aktifasi untuk reaksi yg dikatalis dengan ensim lebihrendah dr reaksi tanpa ensim

Glukosa + 6 O2 6 CO2 + 6 H2O G = -2880 kJ/mol

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Enzim penting untuk menurunkan energi aktifasi untuk

memulai suatu reaksi

Enzim mengikat substrat menciptakan jalan reaksi yg

berbeda yg mempunyai fase transisi lebih rendah dbandingreaksi tanpa enzim

Inti dr reaksi katalisis ikatan yg spesifik pd fase transisi

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Mekanisme kerja enzim

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Fig. 5.5 Enzyme shapes determines its activity

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g y p y

Lysozyme

Changes upon

binding of the

substrate

The substrate is

now bound more

intimately

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Gambar sisi aktif ensim dan asam amino yang

terlibat

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Sisi aktif mempunyai 2 bagian yg penting:

Bagian yang mengenal substrat dan

kemudian mengikatnya

Bagian yang mengkatalisis reaksi, setelah

substrat diikat oleh enzim

Asam amino yang membentuk kedua

bagian tersebut tidak harus berdekatan

dalam urutan secara linear, tetapi dalamkonformasi 3D mereka berdekatan

Teori untuk menjelaskan kerja

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Teori untuk menjelaskan kerja

enzim:

Lock and Key analogyEnzim memiliki struktur sisi spesifik yang cocokdengan substrat.

Mampu menerangkan spesifitas ensim ttp tidak dapat

menerangkan stabilitas fase transisi ensim Induced Fit theory

mempertimbangkan fleksibilitas protein, sehinggapengikatan suatu substrat pada enzim menyebabkansisi aktif mengubah konformasinya sehingga cocok

dgn substratnya. dpt menerangkan fase transisi ESkomplek

Lock and Key

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Lock and Key

http://www.lewport.wnyric.org/jwanamaker/

animations/Enzyme%20activity.html

http://www.lewport.wnyric.org/jwanamaker/animations/Enzyme%20activity.htmlhttp://www.lewport.wnyric.org/jwanamaker/animations/Enzyme%20activity.htmlhttp://www.lewport.wnyric.org/jwanamaker/animations/Enzyme%20activity.htmlhttp://www.lewport.wnyric.org/jwanamaker/animations/Enzyme%20activity.html

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Enzyme activity

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Enzyme activity

Temperature and pH affect the activityof an enzyme.

E d H

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Enzyme Activity and pH

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Optimum Condition

Enzymes function best or are most activein specific conditions known as optimum

conditions.

Kinetika Reaksi Enzimatis

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Kinetika Reaksi Enzimatis

E + S ES E + PK1 K2

K-1

K1 : kecepatan konstan pembentukan ES komplek

K2 : kecepatan konstan konfersi ES komplek ke PK-1 : kecepatan konstan pemecahan ES komplek ke E

bebas

Enzim sangat efisien dalam mengkatalis suatu reaksi,

steady state(keseimbangan reaksi) segera dapat tercapaiapabila : Kecepatan pembentukan ES komplek sama

dengan kecepatan pemecahannya

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K-1 + K2

= Km konstanta MichaelisK2

Vmax [S]V = Persamaan Michaelis-Menten

Km + [S]

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Lineweaver-Burk double reciprocal plot

Y = m x + b

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penghambatan competitive

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penghambatan competitive

inhibitor bersaing dgnsubstrat untuk terikat pdsisi aktif

Biasanya inhibitorberupa senyawa yg

menyerupai substratnya,& mengikat enzimmembentuk komplek EI

krn terikat scrreversible penghambatan nya

bias, yaitu ketikaditambah substrat makapenghambatanberkurang

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Penghambatan un-competitive

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Penghambatan un-competitive

Terikat pd sisi selain sisiaktif enzim

Berbeda dgn non-competitive inhibitor

ini hanya terikat pd ESkomplek

Sehingga tidak terikat pdenzim bebas

Vmax

berubah, dan Kmjuga berubah

E i ll t ik

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Enzim allosterik

Enzim allosterik mengalami perubahan konformasi sebagai respon terhadap pengikatan modulator efektor

Allosterik enzim biasanya lebih komplek dari nonallosterik enzim, memiliki sub unit lebih dari satu

Memiliki satu atau lebih sisi allosterik / regulator untukmengikat modulator.

Seperti halnya substrat, setiap regulator memiliki sisipengikatan yang berbeda

Untuk enzim homotropik sisi aktif dan sisi regulatorsama

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1. Enzim bersifat koloid, luas permukaan besar, bersifathidrofil

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hidrofil

2. Dapat bereaksi dengan senyawa asam maupun basa,kation maupun anion

3. Enzim sangat peka terhadap faktor-faktor yangmenyebabkan denaturasi protein misalnya suhu, pH dll

4. Enzim dapat dipacu maupun dihambat aktifitasnya

5. Enzim merupakan biokatalisator yang dalam jumlah

sedikit memacu laju reaksi tanpa merubahkeseimbangan reaksi

6. Enzim tidak ikut terlibat dalam reaksi, struktur enzimtetap baik sebelum maupun setelah reaksiberlangsung

7. Enzim bermolekul besar

8. Enzim bersifat khas/spesifik

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specific activity is the amount of product formed by an enzyme

http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzyme

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spec c act ty p y yin a given amount of time under given conditions per milligram ofenzyme.

The rate of a reaction is the concentration of substrate

disappearing (or product produced) per unit time (molL 1s1)

The enzyme activity is the moles converted per unit time (rate reaction volume). Enzyme activity is a measure of quantity ofenzyme present. The SI unit is the katal, 1 katal = 1 mol s-1, butthis is an excessively large unit. A more practical value is 1enzyme unit (EU) = 1 mol min-1 ( = micro, x 10-6).

The specific activity is the moles converted per unit time perunit mass of enzyme (enzyme activity / actual mass of enzymepresent). The SI units are katal kg-1, but more practical units aremol mg-1 min-1. Specific activity is a measure of enzyme

efficiency, usually constant for a pure enzyme. If the specific activity of 100% pure enzyme is known, then an

impure sample will have a lower specific activity, allowing purityto be calculated.

The % purity is 100% (specific activity of enzyme sample /specific activity of pure enzyme). The impure sample has lowerspecific activity because some of the mass is not actually

http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzyme

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