1 enzymes. objectives what are enzymes ? properties of enzymes classification factors affecting...

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Page 1: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

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EnzymesEnzymes

Page 2: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

ObjectivesObjectives

• What are enzymes ?

• Properties of enzymes

• Classification• Factors Affecting Enzyme Action• Enzyme Kinetics

Page 3: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

What Are Enzymes?What Are Enzymes?

Page 4: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

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• BiologicalBiological CatalystCatalyst

• Most enzymes are Proteins Proteins

• Not permanentlyNot permanently changed in the process

• Not consumedNot consumed

Page 5: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Differ from inorganic catalysts in:

•Being sensitive to changes in pH , temperature ,

•loss of activity by time •specificity

Page 6: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Definitions•Ribozymes : are RNAs with catalytic activity.

•catalyze the cleavage & synthesis of phosphodiester bonds

•Zymogen•Isozymes

Page 7: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Isoenzymes:• enzymes that catalyze the same

reaction but may have genetically determined difference in the amino acid sequence

•Thus may have difference in their physical properties

Page 8: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

The Active Site

Page 9: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Plasma enzymes 2 major groupsmajor groups:

•1-Functional plasma enzymes• perform specific physiological function.• Examples:• Zymogens (inactive

precursors) of enzymes involved in blood coagulation.

•2-Non-functional plasma enzymes

• no physiological role in the blood. • occur in blood in very minute amounts).

Page 10: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Enzymes composed wholly of protein are known as simple enzymes

• ComplexComplex enzymes, which are composed of protein + small molecule holoenzymes.

• Protein component apoenzyme, while the non-protein component coenzyme or cofactor

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assistance

Page 12: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Prosthetic group describes a complex in which the small organic molecule is permanently bound to the apoenzyme by covalent bonds and cosubstrate occurs when it is transiently attached.

• Coenzymes are often derivatives of vitamins (FAD,NAD, CoA)

Page 13: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Catalytic efficiency:Typically 100-1000 substrate

molecules transformed to product / second ?(by enz.)

Turn over number : is the number of substrate moles converted to product/enzyme mol /sec.

Page 14: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Location within the cell:•Mitochondria: TCA cycleFatty oxidationDecarboxylation•Cytosol:GlycolysisHMPFatty acid synthesis•Lysosomes:Degradation of macromolcule

Page 15: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Nomenclature of Enzymes& Classification

Page 16: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

•Recommended name uricase ,glucosidase

Page 17: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Systematic Name

•Currently enzymes are grouped into six functional classes by the International Union of Biochemistry & Molecular Biology (I.U.B.M.B

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Page 19: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics
Page 20: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

•Regulation:

Rate of product formation responds to the needs of the cells

Page 21: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Factors Affecting Enzyme Action

Page 22: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Temperature

Optimum temperature

ReactionRate

Low High Temperature

Page 23: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Substrate Concentration

Maximum activity

ReactionRate

substrate concentration

Page 24: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

pH:

ReactionRate

Optimum pH

3 5 7 9 11

pH

Narrow range of activityMost lose activity in low or high pH

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Page 26: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• This is because by time the substrate decreases (↓v1 ) ,

• the product increases(↑v-1 ) besides

• the enzyme decreases due to denaturation.

Time

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27

How do enzymes Work?

Enzymes work by

weakening bonds which

lowers activation

energy

Page 28: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Substrate

If enzyme just binds substrate then there will be no further reaction

Transition state Product

Enzyme not only recognizes substrate, but also induces the formation of transition state

X

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Enzymes

FreeEnergy

Progress of the reaction

Reactants

Products

Free energy of activationFree energy of activation

Without Enzyme

With Enzyme

Page 30: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

The active site consists of certain groups e.g.-SH, -OH, COO-, NH3 + & imidazole.

Some groups are concerned with binding of the substrate & that determines the specificity of the enzyme.

Page 31: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Other groups are concerned with catalysis.

• In some enzymes these groups can participate in general acid-base catalysis. In others, catalysis may involve transient formation of a covalent enzyme-substrate complex.

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Page 33: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics
Page 34: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics
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Page 36: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics
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Inhibition of enzyme activity

• InhibitorInhibitor :is any substance that can diminish the velocity of the enzyme catalyzed reaction.

• Competitive

• Non competitive: Heavy metal ions (e.g. mercury and lead) should generally be prevented from coming into contact with enzymes as they usually cause such irreversible inhibition by binding strongly to the amino acid backbone.

• Uncompetitive

Page 38: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Reversible Inhibition:Competitive

Page 39: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Non Competitive Inhibition:

Page 40: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics
Page 41: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics
Page 42: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Ca

rtoo

n G

uid

eEq

uatio

n an

d De

scrip

tion

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S → ES → E + P + II↓EII

E + S → ES → E + P + + II II↓ ↓EII + S →EIIS

↑ ↑

E + S → ES → E + P + II ↓ EIIS

EI

S X

Juang RH (2004) BCbasics

Page 43: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Sulfa Drug Is Competitive Inhibitor

-COOHH2N-

-SONH2H2N-

Pteridine PrecursorFolicacid

Tetrahydro-folic acid

SulfanilamideSulfa drug (anti-inflammation)

Para-aminobenzoic acid (PABA)

Bacteria needs PABA for the biosynthesis of folic acid

Sulfa drugs has similar structure with PABA, andinhibit bacteria growth.

Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197

Page 44: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Regulation of Enzyme Activity

•Substrate availability•Allosteric Effectors•Covalent modification•Induction & repression

Page 45: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Regulation maybe short term regulation or long term regulation

• The long term regulation includes enzyme synthesis & repression

• The short term regulation includes allosteric regulation & covalent modification

Page 46: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Allosteric Effectors

•AllostericAllosteric means (“occupy another space”, other than that of the substrate).

•Allosteric enzymesAllosteric enzymes are enzymes whose activity at the catalytic site may be modulated by the presence of allosteric effectors at an allosteric site (i.e. at a site other than the active site.)

•Allosteric enzymes usually contain multiple subunitsusually contain multiple subunits.•The molecules regulating the allosteric enzymes are called effectors (modifiers or modulatorsmodifiers or modulators).

Page 47: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

• Allosteric effectors bind non-covalentlynon-covalently at a site other than the active site.

• Binding of allosteric effectors at the allosteric site induces conformational changes at the catalytic site & this can alter the affinity of the enzyme for its substrate or modify the maximal catalytic activity of the enzyme or both.

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Covalent Modification

•Addition or removal of phosphate groups from specific serine, threonine, tyrosine

•The phosphorylated form of the enzyme may be activated or deactivated according to the specific enzyme

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Page 53: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Result of Regulation

Page 54: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Blood SampleBlood Sample

Page 55: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Plasma X Serum

Physiological Lab.

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Thank You

Page 57: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

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EnzymesEnzymes•Are specific

for what they will catalyzecatalyze

•Are ReusableReusable

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• Enzymes as indicator tools

• In healthy individuals, levels of these enzymes are almost constant as it reflects a steady state

• elevated enzyme activity in plasma may indicate tissue damage which is accompanied by increased release of intracellular enzymes.

Page 59: 1 Enzymes. Objectives What are enzymes ? Properties of enzymes Classification Factors Affecting Enzyme Action Enzyme Kinetics

Enzyme Stabilizes Transition State

S

P

ES

EST

EP

ST

Reaction direction

Energy change

Energy required (no

catalysis)

Energy decreases (under

catalysis)

T = Transition state

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166