ENZIM

(1)

ENZIM

Oleh:

Maria Ulfah, S.Si, M.Pd

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What Are Enzymes?

 Most enzymes are

Proteins

Proteins (tertiary (

and quaternary structures)

 Act as CatalystCatalyst to

accelerates a reaction

 Not permanentlyNot permanently

(3)

Enzymes

 Are specific for

what they will catalyze

catalyze

 Are ReusableReusable  End in –asease

(4)

Enzyme-Substrate Complex

The substancesubstance

(reactant) an

enzyme

enzyme acts on is the substratesubstrate

Enzyme

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Formation of an enzyme-substrate complex

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How do enzymes

Work?

Enzymes work

by weakening

weakening

bonds

which

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Active Site

 A restricted regionrestricted region of an enzymeenzyme

molecule which bindsbinds to the substratesubstrate

Enzyme

Substrate

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The enzyme active site (features)

 The catalytic site is relatively small compared

with the rest of the enzyme. Why are many enzymes so big then?

 The catalytic site is a three-dimensional entity

 Substrates are bound to enzymes by multiple

weak, non-covalent interactions (electrostatic bonds, hydrogen bonds, van der Waals

(13)

The specificity of binding depends on the

precisely defined arrangement of atoms in

an active site

 Emil Fischer (over

100 years ago): came up with the “lock and key” hypothesis to describe

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(15)

Induced Fit

 A change in

the shapeshape of an enzyme’s active site

 Induced Induced by the

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 “Induced fit” model: a

more refined model that takes into

account the enzyme assumes a

complimentary shape to that of its substrate only after substrate binds to the enzyme.

 More dynamic

scenario compared to the lock and key

(17)

 A change in the configurationchange configuration of an enzyme’s active

enzyme’s active site (H+ and ionic site

bonds are involved)

 Induced by the substrateInduced substrate

substrate

Enzyme Active Site

induced fit

Induced Fit

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What Affects Enzyme Activity?

What Affects Enzyme Activity?

 Three factors:Three factors:

1.

1. Environmental ConditionsEnvironmental Conditions 2.

2. Cofactors and CoenzymesCofactors and Coenzymes 3.

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1. Environmental Conditions

1. Extreme Temperature1. Extreme Temperature are the most are the most

dangerousdangerous

- high temps- high temps may denature (unfold) denature (unfold) the enzyme.enzyme.

2.

2. pHpH (most like 6 - 8 pH near neutral) (most like 6 - 8 pH near neutral) 3.

(20)

Environmental factors affecting enzyme activity

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2. Cofactors and Coenzymes

 Inorganic substances (zinc, iron)Inorganic substances (zinc, iron)

and vitaminsvitamins (respectively) are sometimes need for proper

enzymatic activity

enzymatic activity.

 Example:Example:

Iron

Iron must be present in the

quaternary

quaternary structure structure -

-hemoglobin

hemoglobin in order for it to pick up oxygen.

(22)

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Michaelis-Menten model of

enzyme kinetics (Vmax & Km)

 Key element in their model is the

existence of the ES complex

 Rate of catalysis (V) increases with

(24)

 When enzyme concentrations are constant, V is

linearly proportional to [S] WHEN [S] IS SMALL.

 At high [S] (when S is in vast excess of the

(25)

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Km & Vmax

 Km = the Michaelis constant

 Defined as the [substrate] at which the

reaction rate is half of its maximal value

 Used to define relative affinity of an

enzyme for its substrate

 The higher the Km value, the lower the

(27)

 Vmax: describes the maximal rate of product formation when [S] is high

 Under such conditions all of the existing “pool” of enzyme active sites are full

 From Vmax an enzyme’s turnover

(28)

Double-reciprocal

(lineweaver-Burk) plot

 Used to calculate Km & Vmax

 Also used to characterize

mechanisms of enzyme inhibition by specific

compounds

 Data expressed as 1/V

(29)

(30)

Allosteric enzymes do not conform

to Michaelis-Menten kinetics

 Yield a sigmoidal curve on a V versus S

plot (not hyperbolic as seen under Michaelis-Menten conditions)

 Sigmoidal curve indicates cooperative

binding (binding of one molecule of S affects affinity and binding of

additional S molecules)

 Regulatory molecules can alter

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Enzyme inhibition

 For enzymes that obey Michaelis-Menten

laws, compounds that reversibly inhibit enzyme activity can be kinetically

classified

 Consider two general types:

Competitive inhibitors

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Competitive inhibitors:Competitive inhibitors: are chemicals that resembleresemble an

enzyme’s normal substrate

enzyme’s normal substrate and

compete

compete with it for the active site.active site

Enzyme

Competitive inhibitor

(35)

Noncompetitive inhibitors:Noncompetitive inhibitors:

Inhibitors that do not enter the do not enter the

active site

active site, but bind to another bind to another part

part of the enzyme causing the enzyme enzyme

enzyme to change its shape, which change its shape

in turn alters the active site.lters the active site

(36)

Competitive inhibitors

 Y intercept the same regardless of whether

(37)

 Do not alter Vmax  Increase Km

 Competitive inhibition can be overcome by

increasing substrate concentration

 Block substrate binding to the active site

of an enzyme

(38)

Examples of competitive inhibitors

 Alcohol (alcohol dehydrogenase)

 UpCA (RNase)

 DHFR inhibitors (DNA metabolic inhibitor of

tumors)

 Sulfa drugs (anti-bacterial drugs)

 Physiological examples: feedback

(39)

Enzyme inhibition & automobile

antifreeze

 Ethylene glycol (EG) is a constituent of

antifreeze

 EG not toxic but is converted to oxalic acid

which form crystals in the kidneys leading to extensive tissue damage and renal

(40)

 First step of conversion of EG to oxalic

acid is its oxidation to an aldehyde by alcohol dehydrogenase

 This reaction inhibited by ethanol which

(41)

An example of a typical competitive inhibitor:

UpCA has a very similar structure to the genuine substrate, but is chemically unable to undergo reaction.

(42)

Folate (folic acid)

Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:

Competitive inhibitors of dihydrofolate reductase used in cancer treatment (resemble folate, bind ~1000x tighter):

eventually leads to synthesis of thymine nucleotides (DNA metabolism)

(43)

Sulfa Drugs

 Resemble PABA in

structure

 Blocks metabolic

(44)

Examples of the Physiological (regulatory) Role of Enzyme Inhibitors

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Another example of regulatory competitive inhibition: Inhibition

(46)

Noncompetitive inhibitors

 Plots converge on the X axis in the

(47)

Noncompetitive inhibitors

 Do not alter Km  Decrease Vmax

 Noncompetitive inhibition cannot be

overcome by adding excess substrate

 Bind to a site outside of catalytic site of

(48)

In noncompetitive inhibition

(49)

The inhibitor lowers the concentration of functional enzyme

 The remaining “uninhibited” enzyme behaves

like a more dilute solution of that enzyme (assumes [inhibitor] is limiting)

 In other words, the substrate can still bind to

enzyme alone or enzyme complexed with the inhibitor. But only free enzyme will catalyze the reaction.

 Since the pool of free enzyme is lower in

(50)

Irreversible Enzyme Inhibitors

 Inhibitor becomes covalently linked to the

enzyme

 Attachment often occurs at the active site  Examples: 5-fluorouracil, DIPF (nerve

(51)

Suicide Inhibitors

 Irreversible enzyme inhibitors

 Participate in the enzymatic reaction like the

substrate

 At some point in the reaction they get ‘stuck’

and become permanently linked to the enzyme.

 Example: 5-Fluorouracil, a suicide inhibitor

(52)

5Fluorouracil TS cannot catalyze

(53)

Enzyme inhibitors as anti-bacterial drugs

(54)

Most Drugs and

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Enzyme Inhibition – 1: Irreversible

 Denaturation or

specific sites

 Heavy metals

 Usually try to avoid

 EDTA in buffers

 Occasionally useful

experimentally

 Hg-inactivation can

implicate Cys

 If bind at active site:

 Substrate can

reduce rate of

irreversible inhibition

 Rate(inhibition) vs.

[S] gives substrate dissociation

constant, KS.

(56)

Reversible inhibition

 Competitive, non-competitive, mixed,

uncompetitive

 Each suggestive of a different mechanism  Actually, operational definitions

Depend on kinetic behavior

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1. Competitive inhibition - 1

 Poss. Mechanism: Binds in active site,

competing with substrate.

S + E + I

ES EI

K_i Inhibition constant: in this _{mechanism, this is a true}

dissociation constant, because EI is a “dead-end” complex

K_M (apparent

(58)

Competitive inhibition 2 - example

 Succinate dehydrogenase

CO₂

-CH₂ CH₂ CO₂

-Succinate

CO₂

-CH

CH

CO₂

-Fumarate CO₂

-CH₂ CO₂

(59)

Competitive inhibition 3: kinetics

 Kinetics in the presence of many types of inhibitor

looks like Michaelis-Menton w/ modified constants:

 With competitive inhibition...

 

S ; S max   _app M app K V v

 





 

max max

max ; S I 1 S V V K K V v app i M     app M

(60)

Competitive inhibition 4 - plots

 Kinetics show V_max

unchanged

Non-mathematically - if [S] >> [I], drown out inhibition

 Affect (increase) K_M

amount of S required for 1/2 Vmax

1/v₀ Slope = K_M/V_max

(61)

Competitive inhibition 5: mechanism

 Previously suggested mechanism was one of

several that lead to similar kinetic effects

 Perhaps I is really an alternative substrate

 Competitive inhibition defined operationally

 Often don’t know the mechanism

 Any inhibitor that affects K_M, but not V_max

 The “specificity” constant = k_cat/K_M is changed

 Some use “specific inhibition” to avoid mechanistic

(62)

2. Non-competitive inhibition

 Operational definition

 Lower V_max, without changing K_M.

 affect rate w/o affect substrate-binding

 Possible mechanisms

 Very small addition to active site

 e.g. H+ or cation.

 Remote inhibitor binding that affects exact positioning of catalytic

groups

 Difficult to distinguish from irreversible inhibition

 V_max = k_cat[E₀] can be lowered by

 reducing k_cat - non-competitive inhibition

 reducing amount of active enzyme - irreversible inhibition

 Rare: difficult to affect k_cat w/o affecting K_M.

 More commonly, component of “mixed inhibition”.

(63)

3. Mixed inhibition

 Operational definition

 Lower V_max,

 also raise K_M, EIS more likely to dissociate than ES

1/v₀

1/V_max -1/K_M

inhi bite

d

(64)

4. Uncompetitive inhibition

 Operational: Km and Vmax are

changed by the same factor.

 Possible mechanism: Inhibitor

binds to ES complex, not at all to free-E.

 Naturally rare:

 Inhibition of myo-inositol

monophosphatase by Li+

anti-depressant

 Experimental product inhibition

often uncompetitive

1/v₀

1/V_max -1/K_M

inhibi te

d

(65)

Classification of Inhibitors according to

effect on kinetic parameters

Vapp_/

KMapp

Vapp

Competitive Yes No i.e. only K_M changed

Uncompetitive No Yes V_max & K_M changed by corresponding amounts giving / / double- reciprocal plots

(66)

Enzymes

Free

Energy

Progress of the reaction

Reactants

Products

Free energy of activation

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Catalytic sites form clefts or

crevices

 Substrate molecules bound within cleft

 Water (unless involved in catalysis) is

normally excluded

 Overall nonpolar character of cleft can

enhance binding of substrate

 Cleft may also contain polar residues which