dr.Syahrijuita, M.Kes, Sp.THT-KL Deparment of Biochemistry

Medical Faculty of Hasanuddin University

ENZYMESENZYMES

• Definition,composition, structure and properties of enzyme• How Enzymes work• Enzyme activity• Factors affecting enzyme activity• Regulation of enzyme activities• Enzymes in clinical diagnosis

Topic

Definition of enzyme •Enzymes are biological catalysts.•A Catalyst is defined as "a substance that increases the rate of a chemical reaction without being itself changed in the process.”

INTRODUCTION Enzymes are biological catalysis

- substance of biological origin that accelerate chemical reactions.- the vast majority of enzyme are proteins.- catalytically active ribonucleic acids “ ribozymes”. - in general, name suffix “-ase”

The presence and maintenance of a complete and balanced set of enzymes is essential for - the breakdown of nutrients to supply energy, and chemical building block; - the assembly of those building blocks into proteins, DNA, membranes, cells, and tissues; - and the harnessing of energy to power cell motility and muscle contraction.

Deficiency in quantity and catalytic activity can result from:- genetic defects,- nutritional deficits,- or, toxins.

Defective enzymes can result from: - genetic mutation- infections by viral or bacterial pathogens.

Medical scientists:- imbalances in enzyme activity

pharmacologic agent to inhibit.- investigating gene therapy

remedy deficits in enzyme level and function.

Enzymes as Biological CatalystsEnzymes are

proteins that increase the rate of reaction by lowering the energy of activation

They catalyze nearly all the chemical reactions taking place in the cells of the body

Enzymes have unique three-dimensional shapes that fit the shapes of reactants (substrates)

Properties of enzymes (important!)

•Catalytic efficiency – high efficiency, 103 to 1017 faster than the corresponding uncatalyzed reactions

•Specificity - high specificity, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction.

•Mild reaction conditions- 37℃, physiological pH, ambient atmospheric pressure

Chemical composition of enzymes

(1) Simple protein

(2) Conjugated protein

Holoenzyme= Apoenzyme+ Cofactor

Coenzyme : loosely bound to enzyme (non-covalently bound).

Prosthetic group : very tightly or even covalently bound to enzyme (covalently bound)

Cofactor

Classification of enzymes

1). Monomeric enzyme2). Oligomeric enzyme

3). Multienzyme complex: such as Fatty acid synthase

(1). By their composition

(2) Nomenclature • Recommended name

•Enzymes are usually named according to the reaction they carry out.

•To generate the name of an enzyme, the suffix -ase is added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers).

•Systematic name (International classification)

• By the reactions they catalyze (Six classes)

Enzyme Nomenclature International Union of Biochemist (IUB)In general:

- Type of reaction catalyzed followed by suffix –- Type of reaction catalyzed followed by suffix –asease..- dehydrogenase, protease, etc.

IUB:IUB:- Each enzyme has a unique name and code number that reflect the type of - Each enzyme has a unique name and code number that reflect the type of reaction catalyzed and the substrate involved.reaction catalyzed and the substrate involved.- EC 1.2.3.4- EC 1.2.3.4

- EC, Enzyme catalog- EC, Enzyme catalog- 1, Class- 1, Class- 2, subclasses - 2, subclasses - 3, subsubclasses- 3, subsubclasses- 4, where the enzyme belongs in the subsubclases.- 4, where the enzyme belongs in the subsubclases.

- - EC 2.7.1.1- class 2, transferase- class 2, transferase- subclass 7, transfer of a phosphoryl group.- subclass 7, transfer of a phosphoryl group.- subsubclass 1, alcohol is the phosphoryl acceptor.- subsubclass 1, alcohol is the phosphoryl acceptor.- 1, hexose-6, alcohol phosphorylated is the of carbon-6 of a - 1, hexose-6, alcohol phosphorylated is the of carbon-6 of a

hexose.hexose.

Enzyme Classes (IUB)

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5. How enzymes work (important!)1) Enzymes lower a

reaction’s activation energyAll chemical reactions

have an energy barrier, called the activation energy, separating the reactants and the products.

activation energy: amount of energy needed to disrupt stable molecule so that reaction can take place.

Enzymes Lower a Reaction’s Activation Energy

What is the difference between an enzyme and a protein?

Protein

•All enzymes are proteins except some RNAs • not all proteins are enzymes

RNAEnzymes

2) The active site of the enzyme Enzymes bind substrates to their active

site and stabilize the transition state of the reaction.

The active site of the enzyme is the place where the substrate binds and at which catalysis occurs.

The active site binds the substrate, forming an enzyme-substrate(ES) complex.

Active siteBinding site

Catalytic site

Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate© 2007 Paul Billiet ODWS

The Lock and Key HypothesisThis explains enzyme specificityThis explains the loss of activity when

enzymes denature

© 2007 Paul Billiet ODWS

Induced Fit ModelEnzymes can form to the shape of its

substrate.

http://en.wikipedia.org/wiki/File:Induced_fit_diagram.svg

Life Sciences-HHMI Outreach. Copyright 2009 President and Fellows of Harvard College

The Induced Fit Hypothesis

This explains the enzymes that can react with a range of substrates of similar types

Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

© 2007 Paul Billiet ODWS

1. Substrate approaches active site2. Enzyme-substrate complex forms3. Substrate transformed into products4. Products released5. Enzyme recycled

6. Enzyme activityEnzymes are never expressed in terms of their

concentration (as mg or μg etc.), but are expressed only as activities.

Enzyme activity = moles of substrate converted to product per unit time.The rate of appearance of product or the rate of

disappearance of substrateTest the absorbance: spectrophotometer

7. Factors affecting enzyme activityConcentration of substrateConcentration of enzymeTemperaturepHActivatorsInhibitors

Enzyme velocityEnzyme activity is commonly expressed by the

intial rate (V0) of the reaction being catalyzed. (why?)

• Enzyme activity = moles of substrate converted to product per unit time.

Note: V means V0[S]

[S] + KMV = Vmax

Km: Michaelis constantKm = (k2 + k3)/k1

1. Michaelis-Menten equation describes how reaction velocity (V) varies with substrate concentration [S].

• The following equation is obtained after suitable algebraic manipulation.

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Persamaan Michaelis-Menten

Vomax [S]Vo= ---------- Km + [S]

Apabila [S] <<< Km

[S] >>> Km

[S] = Km

[S]>>[E] V∝[E] •The initial rate of an enzyme-catalyzed reaction is always proportionate to the concentration of enzyme.

•This property of enzyme is made use in determining the serum enzyme for the diagnosis of diseases.

(2) Effect of [E] on velocity

(3) Effect of temperature on velocity

Bell-shaped curve

(4) Effect of pH value on velocity• Each enzyme has an optimal pH or pH range (where the enzyme has maximal activity).• Requirements for the catalytic groups in the active site in appropriate ionization state is a common reason for this phenomenon.

•The pH optimum varies for different enzymes.•Most enzyme: neutral pH (6-8).

Bell-shaped curve

Optimum pH values

Enzyme activity Trypsin

Pepsin

pH1 3 5 7 9 11

© 2007 Paul Billiet ODWS

(5) Effect of activator on velocity

(i). Inorganic ions • Metal ions ， such as Na+, K+, Mg2+, Ca2+, Cu2+, Zn2+, Fe2+ et al

• Anions: such as Cl-, Br-, I- 、 CN- et al

(ii). Organic

(iii). Proteins

• Reducing agents, such as Cys 、 GSH

•Enzyme activators are molecules that bind to enzymes and increase their activity.

(6) Inhibition of enzyme activities(very important!)

• Inhibitor: any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor.(not denaturation)

• Some enzyme inhibitors are normal body metabolites.

• Other may be foreign substances,such as drugs or toxins.

8. REGULATION OF ENZYME ACTIVITY

1. Allosteric binding sites: Allosteric enzymes are regulated by molecules called effectors (modifiers) that binds nonconvalently at a site other than the active site.

2. By Covalent Modification: Many enzymes are regulated by covalent modification, most frequently by the addition or removal of ‘phosphate’ group to serine, threonine or tyrosine residue of the enzyme by kinases. (enzyme)

3. Induction and repression of enzyme sysnthesis: Cells can also regulate the amount of enzymes present by altering the rate of enzyme synthesis.

REGULATION CONT….4. Zymogen Cleavage: Some enzyme are

synthesized as inactive precursor, called zymogens, that are activated by proteolysis (e.g., digestive enzyme, pepsinogen is inactive and cleaved to pepsin which is active chymotrypsin)

5.Location within the cell: Many enzymes are localized in specific organelles within the cell. This, compartmentation helps in the regulation of the metabolic pathway.

9. Enzymes in clinical diagnosisAn enzyme test is a blood test or urine test

that measures levels of certain enzymes to assess how well the body’s systems are functioning and whether there has been any tissue damage. (why?)

Principal Serum Enzymes Used in Clinical Diagnosis.

Note: Many of the enzymes are not specific for the disease listed.

Some diseases caused by enzyme malfunctions

diseasesdiseases Enzyme malfunctionEnzyme malfunction

AlbinoAlbinoAlkaptonuriaAlkaptonuriaGalactosemiaGalactosemiaHomosistinuriaHomosistinuriaphenylketonuriaphenylketonuriaTay-sachsTay-sachs

3-monooksigenase tyrosine3-monooksigenase tyrosine1,2-dioksigenase homogentisat1,2-dioksigenase homogentisatUridilil transferase galactosa 1-fosfatUridilil transferase galactosa 1-fosfat-sintase sintationine-sintase sintationine4-monooksigenase phenylalanine4-monooksigenase phenylalanineHeksosaminidase AHeksosaminidase A

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http://www.chem.qmul.ac.uk/iubmb/enzyme/