1 properties of enzymes 2014

7/21/2019 1 Properties of Enzymes 2014

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Enzymes

Biological catalysts Most are proteins (except a small group of catalytic RNAmolecules)

increase rates of a chemical reaction

do not effect equilibrium of reactions

remain unchanged in overall process reactants (substrates) bind to enzymes, products are released

selectively recognize proper substrates over other molecules

Accelerate reaction rates tremendously over un-catalyzedrates


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Distribution of all known enzymes byclassification:

- Enzymes are usually named for the substrates and type of reactionwith the suffix -ase.


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1. Oxidoreductases

2. Transferases


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3. Hydrolases

4. Lyases


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5. Isomerases

6. Ligases


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Enzyme kinetics

- Initial velocities are used to indicate reaction rates

- Study of the rates of enzyme-catalyzed reactions under differentconditions.

Why do the curves flatten over time?

S P


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Reaction rate does not increase linearly with substrate conc.:

HyperbolicKinetics

In enzyme-catalyzed reactions, velocity is usually proportional to enzymeconcentration [E]:


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A P v = k[A] where k = rate constant

Rate equations for chemical reactions

First order reactionk

A + B P v = k[A] [B]

Second order reaction

What about enzyme-catalyzed reactions?

Simple case: single substrate (unimolecular) reaction

ES+ E S E + P

E + S ES E + Pk1

k-1

k2

k-2

k


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The Michaelis and Menten equation explains the hyperbolic kinetics

The M-M equation:

The equation for a rectangular hyperbola:

Km(Michaelis constant) = (k-1+ k2)/k1

E + S ES E + Pk1

k-1

k2

k-2


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E + S ES E + Pk1

k-1

k2

k-2

- Velocity at time zero (initial velocity) v0= k2[ES];

How was the M-M equation derived?

Several assumptions were made when deriving the M-M equation

1. Early in the reaction (close to time zero), there is no ES formation from E + P

because [P] is negligible, hence k-2can be ignored.

E + S ES E + Pk1

k-1

k2

- [ES] is very difficult to determine


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2. Steady state assumption for enzyme-substrate complex concentration[ES]

Rate of ES formation = Rate of ES breakdown

k1[E] [S] = k-1[ES] + k2[ES]

since [E] = [E]total

[ES] , thenk

1([E]total

[ES]) [S] =k

-1[ES] +k

2[ES]

E + S ES E + P

k1

k-1

k2

ET ES [S]

[ES]=

1+2

1

Km

(Michaelis constant) =1+2

1

Dividing both sides by [ES] and k1:


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ET ES [S]

[ES]= Km Solving for [ES]: [ES] =

ET S

m

+ [S]

Velocity at time zero (initial velocity) v0= k2[ES];

E + S ES E + P

k1

k-1

k2

v0=

2

ET S

m+ [S]

3. Maximum reaction velocity occurs when enzyme is saturated with substrate

When [S] is very high, v0= Vmaxand [ES] = [E]T (all enzymes are present as ES)

So, Vmax= k2[ES] = k2[E]T

Substituting Vmax = k2[E]Tinto the above equation:

v0 =

max

S

m+ [S] The M-M equation


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The Michaelis constant (Km)

- When v0is one half of Vmax, the M-M equation becomes

- therefore, Km= substrate conc. at which V = Vmax

-K

mcan be determined experimentally


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- k2 is much smaller than k1or k-1when k2is rate-limiting (as is often thecase).

- Then, k2can be neglected and Km=1

1

(Dissociation constant for ES)

- Therefore Km is a measure of the affinity of E for S

- Small Kmmeans higher affinity of the enzyme for a substrate (ES complexmore stable)

- high Kmmeans lower affinity of the enzyme for a substrate (ES complexless stable)

The Michaelis constant (Km) (continued)

Km(Michaelis constant) =1+2

1

E + S ES E + Pk1

k-1

k2


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- Kmis unique for each enzyme-substrate pair

The Michaelis constant (Km) (continued)


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The catalytic constant (Kcat)- At saturating substrate concentration, the velocity is Vmax- Under such condition, reaction rate is determined by enzyme concentration

and the rate constant is called the catalytic constant (Kcat)

Vmax= kcat[E]totalkcat= Vmax /[E]total

- Kcat(s-1) represents the number of moles of substrates converted toproduct per second per mole of enzyme

- i.e. how quickly an enzyme can catalyze the reaction (ES to E + P)- the enzymes turnover number


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- a measure of enzyme effectiveness at low substrate concentration (often thecase under physiological conditions)

v0 =

max S

m

+ [S]

M-M equation:

Vmax= kcat[E]total v0 =

catE

total[S]

m

+ [S]

- when [S] is low (


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Upper limit for kcat/Km

- 108to 109M-1s-1

- Diffusion-controlled limit- Products are formed as soon as the ES complex is formed

The kcat/Km ratio (Enzymatic rate constant) (continued)

Enzyme for whichk

cat/K

m is close to the diffusion-controlled limit


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The Catalytic Proficiency= enzymatic rate constant/non enzymatic rate constant


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How do you get the values for Vmax, Kmand kcat?

- Kmand Vmaxvalues can be determined experimentally bymeasurement of initial velocities at a series of substrateconcentrations and a fixed enzyme concentration.

- Kcatvalues can be determined if Vmaxis known and the absoluteconcentration of enzyme is known: kcat= Vmax /[E]total

- Experimentally it is difficult to achieve the substrate conc forVmax determination


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The Lineweaver-BurkePlot (double reciprocal plot)

Plotting 1/[S] vs 1/Vo

L-B equation for straight line

Easier to extrapolate values w/straight line vs hyperbolic curve

Y-intercept = 1/VmaxX-intercept = -1/Km

M-M equation:

(y = mx + c)


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Multisubstrate reactions

A + B P + QE

Common kinetic mechanisms:


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Enzyme Inhibition

Inhibitors compounds that bind to an enzyme and interfereswith its activity

Can prevent formation of ES complex or prevent ES breakdownto E + P.

Irreversible inhibitor binds to enzyme through covalent bonds(binds irreversibly)

Reversible Inhibitors bind through non-covalent interactions(disassociates from enzyme)

Why are enzyme inhibitors important?


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(1) Reversible Inhibitors


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Classical competitive inhibitors look like substrates

Example:


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Competitive Inhibitors (CI)

CI binds free enzyme

Competes with substrate for enzyme binding.

Raises Kmwithout effecting VmaxCan relieve inhibition with higher [S] Kmapp= apparent Km


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Uncompetitive Inhibitors


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Uncompetitive Inhibitor (UI)

UI binds ES complexPrevents ES from proceeding to E + P or back to E + S.

Lowers Km& Vmax, but ratio of Km/Vmaxremains the same

1/Vmax

-1/Km


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Noncompetitive inhibitors


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Non-competitive Inhibitor (NI)

NI can bind free E or ES complex

Lowers Vmax, but Kmremains the same

NIs dont bind to S binding site therefore dont effect KmAlters conformation of enzyme to effect catalysis but not substratebinding

1/Vmax

-1/Km

I ibl I hibit


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Irreversible Inhibitors- Form stable covalent bond with enzymes- Active site modification- Permanent inactivation of enzyme activities

Example: DFP (nerve gas)- Organophosphate compound- Inhibitor of hydrolases (e.g. chymotrypsin;

acetylcholine esterase) containing serine aspart of the active site

- Toxic poison developed for military use- Causes paralysis in mammals

SarinThe Syrian government was accused of

using sarin gas in 2013


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Regulation of Enzyme Activity

Enzyme quantity (Response time = minutes to hours)a) Transcription

b) Translation

c) Enzyme degradation

Enzyme activity (rapid response time = fraction of seconds)

a) Allosteric regulation

b) Covalent modification

Enzymes whose activities are under control: Regulated Enzymes

All t i R l ti


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Allosteric Regulation Allosteric enzymes usually have multiple subunits

Allosteric modulators/effectors (inhibitors or activators) bind non-covalently to a regulatory site other than the active site

Regulatory sites in allosteric enzymes may be removed without affectcatalytic functions

Allosteric effectors are not altered chemically by the enzyme

Allosteric effectors do not assemble substrates or products of theenzyme

End products are often allosteric inhibitors feedback inhibition:

B A C13

32

E F G4 5

H I J

4 5

XX

Catalytic and regulatory sites in an allosteric enzyme


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Catalytic and regulatory sites in an allosteric enzyme

Conformation change

What may happen when a negative modulator binds an allosteric enzyme?

- Non-classical competitive inhibitor- Noncompetitive inhibitor

R state (relaxed)

T state (tense)


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Effects of allosteric modulators on enzyme activities

(sigmoidal curve cooperativebinding of substrate)

E l ll t i ti ti d i hibiti f h h f t ki 1


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Example: allosteric activation and inhibition of phosphofructosekinase-1(PFK-1)

Negative modulator: Phosphoenolpyruvate (feedback inhibition)

Positive modulator: ADP

PFK1 catalyzes an early step in glycolysis (an ATP-generating pathway)

Allosteric modulators bind to site other than the active


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Allosteric modulators bind to site other than the activesite and allosteric enzymes have quanternary structure

Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate+ ADP

ADP

PFK-1

Mature enzyme: TetramerSingle subunit Active site

regulatory site

Binding of phosphoenolpyruvate to regulatorysite- Causes PFK-1 to switch to the T state- Lowers PFK-1 affinity for F6P

Binding of ADP to regulatory site- Maintains PFK-1 in the R state- Increases PFK-1 affinity for F6P

(F6P)

R l ti b l t m difi ti


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Regulation by covalent modificationUsually slower than allosteric regulation

Reversible

Require one enzyme for activation and one enzyme for inactivationPhosphorylation/dephosphorylation most common covalent modification

Ser/Thrprotein kinase

Tyrosineprotein kinase

H2OPO42-

Ser/Thrprotein

phosphataseH2OPO4

2-

Tyrosineprotein

phosphatase

H2OPO

42-

E l t d h d ( ti l l i


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- involve protein kinases/phosphatase

- PDK inactivated by phosphorylation

- phosphates are bulky negatively charged groups which affect conformation

h b d b h h l

Example: pyruvate dehydrogenase (connecting glycolysisto the TCA cycle)

serineresidue

1 properties of enzymes 2014

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