Enzimas 2015 01 BqaBasica

BIOQUIMICA BASICA Sem 01/2015

Claudia Rubiano

ENZYMES

Overview

1 Part I: Introduction to enzymes

2 Part II: Enzyme kineticsMichaelis & Menten KineticsAllosteric enzymes kinetics

3 Inhibition of Enzyme ActivityRegulation of enzyme activity

4 Coenzymes

5 Defective enzymes and disease

6 Catalytic RNA




4 Coenzymes


6 Catalytic RNA

KEY CONCEPTS

•  Enzymes differ from simple chemical catalysis in their efficiency and specificity.

•  An enzyme’s name may reflect the reaction it catalyzes.

There are three ways to increase the rate of a chemical reaction.

•  Increasing the temperature – Adding energy in the form of heat

•  Increasing the concentration of the reacting substances

•  Adding a catalyst – A substance that participates in the reaction

yet emerges from the reaction in its original form

Enzymes are catalysts.

•  An enzyme is usually a protein that acts as a catalyst to speed up the rate of a chemical reaction without being consumed itself. – Major exception: ribozymes are RNA

molecules that act as catalysts.

Enzymes speed up biochemical reactions.

Rate enhancements of 108 to 1012 are typical of enzymes.

Enzymes have an active site.

•  Substrates bind to enzymes at the “active site”.

•  Serine proteases are an exemplary class of enzymes that have a common set of amino acids (red) in their active site.

Most enzymes are highly specific for their substrates.

•  Chymotrypsin has broad substrate specificity, though.

•  Chymotrypsin

–  Hydrolyzes peptide bonds after Phe, Tyr, or Trp

–  Hydrolyzes other amide or ester bonds after Phe, Tyr or Trp

Enzymes are usually named after the reaction they catalyze.

•  Pyruvate decarboxylase removes a carboxylate group from the substrate pyruvate.

There are six major classifications of enzymes.

Classification is based on the type of reaction

catalyzed by the enzyme.

What is the enzyme classification for pyruvate decarboxylase?

Answer: Lyase A CO2 group is eliminated

and a double bond is formed to produce CO2.

For more info: see http://enzyme.expasy.org

KEY CONCEPTS

•  The height of the activation energy barrier determines the rate of a reaction.

•  An enzyme provides a lower-energy pathway from reactants to products.

•  Enzymes accelerate chemical reactions using acid-base catalysis, covalent catalysis, and metal ion catalysis.

The height of the activation energy barrier determines the rate of a

reaction.

Products The higher the

activation energy barrier,

the less likely the reaction is to occur!

The sign of !G indicates the spontaneity of a reaction.

!G<0 spontaneous reaction !G>0

non-spontaneous reaction

Enzymes work by lowering the activation energy for a reaction.

Enzymes often use cofactors to aid in catalysis.

There are three fundamental mechanisms for enzyme catalysis.

•  Acid-base – Note: an enzyme can use acid catalysis, base

catalysis or both!

•  Covalent catalysis – Also called nucleophilic catalysis

•  Metal ion catalysis

Some amino acids can play a role in acid-base catalysis.

Covalent Catalysis

•  Covalent catalysts accelerate reactions by forming a covalent bond between E and S.

Nucleophiles & Electrophiles

Electron pair donor or negative charge

Electron-deficient atoms

Metal Ions as Catalysts

Example: Alcohol Dehydrogenase




4 Coenzymes


6 Catalytic RNA

KEY CONCEPTS

• An enzyme’s activity, measured as the rate of product formation, varies with the substrate concentration.

Rates of Chemical Reactions• Enzyme kinetics is the study of rates of

reactions catalyzed by enzymes.

v = S Pk

• The reaction rate (velocity, v) can be described in several ways.– Disappearance of substrate, S– Appearance of product, P

• These equations relate velocity to concentration of reactants and products.

Consider the reaction catalyzed by triose phosphate isomerase.

• As [GAP] decreases, the [DHAP] increases.

GAP DHAP

Reaction velocity can be thought of as concentration vs. time.

Many enzymes react with substrates in a nonlinear fashion.

• The shape here is hyperbolic.

• Shape indicates, in part, that E and S combine to form an ES complex

E + S ES




4 Coenzymes


6 Catalytic RNA

KEY CONCEPTS

• Simple chemical reactions are described in terms of rate constants.

• The Michaelis-Menten equation describes enzyme-catalyzed reactions in terms of KM and Vmax.

Rate equations describe chemical processes.

• A unimolecular reaction has a velocity (rate) that is dependent on the concentration of only one substrate.

• v = k [A], where k has units of sec-1

v = A Pk

Rate equations describe chemical processes.

• A bimolecular (second order) reaction has a velocity (rate) that is dependent on two substrate concentrations.

• v = k [A] [B] (or k [A]2 or k [B]2)where k has units of M-1 ! sec-1

v = A + B Pk

Many enzymes obey Michaelis-Menten kinetics.

E + S ES E + Pk1

k-1

k2

Rate limiting step

Problem:[ES] is difficult to measure!

What can we do?

Try to re-express the rate.

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

Depletion of ESFormation of ES

E + S ES E + Pk1

k-1

k2

One point that helps:

Assume steady state equilibrium.• For most of the duration of the reaction,

[ES] remains steady as substrate is converted to product.

Michaelis-Menten Equation

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

E + S ES E + Pk1

k-1

k2

Derivation of the Michaelis-Menten Equation

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

Thus: k1 [E] [S] = [ES] (k-1 + k2 )

[E]total = [ES] + [E]

[E] = [E]total - [ES]

Since!

Substitute here


Divide both sides by k1

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

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

KM

[E]total [S] - [ES] [S] = [ES] KM


Rearrange:

[E]total [S] - [ES] [S] = [ES] KM

[E]total [S] = [ES] (KM + [S])

[ES] = [E]total [S]

KM + [S]


k2 [E]total [S]

KM + [S]v =

Vmax [S]

KM + [S]v = The Michaelis-

Menten Equation

Vmax

The Michaelis-Menten equation is hyperbolic.

Vmax is where the reaction velocity reaches its plateau

KM is the substrate concentration at ! Vmax

KEY CONCEPTS

• The kinetic parameters KM , kcat, and kcat/KM are experimentally determined.

• K0.5 and Vmax values can be derived for enzymes that do not follow the Michaelis-Menten model.

Kinetic parameters are used to compare enzyme activities

• The Km can vary greatly from enzyme to enzyme, and even for different substrates of the same enzyme:

Kinetic parameters are used to compare enzyme activities

• Considerations about Km and Vmax meaning: They provide little information about the number, rates, or chemical nature of discrete steps in the reaction.

• ONLY UNDER CERTAIN CONSIDERATIONS, Km can be taken as a measure of the affinity of an enzyme for its substrate in the complex ES

Comparing Catalytic Mechanisms and Efficiencies

• Kcat or turnover number: It is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate.

The catalytic rate constant determines how quickly an enzyme can act.

• kcat = catalytic rate constant, turnover

• kcat = k2

• kcat = Vmax/[E]total

Comparing Catalytic Mechanisms and Efficiencies

• Kcat and Km allow to evaluate the kinetic efficiency of enzymes, but either parameter alone is insufficient for this task.

• The best way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to compare the ratio Kcat/Km for the two reactions: Specificity constant

kcat/KM indicates catalytic efficiency.

• What limits the catalytic power of an enzyme?– Electronic rearrangements during formation of the

transition state

– Frequency of productive enzyme collision with substrate, with the maximum being the diffusion-controlled limit

– Enzymes reach catalytic perfection when their rate is diffusion-controlled.

There are algebraical transformations of the MM equation into versions that are useful in the practical determination of Km and

Vmax

The Lineweaver-Burk plot

Vmax [S]

KM + [S]v =

Take the reciprocal of both sides:

v1

= KM

Vmax

+ [S]1

Vmax

1

The Lineweaver-Burk plot

Notice how the data are weighted heavily here due to the linearization!

The EadieHofstee plot

The Hanes-Woolf plot

Many enzymes catalyze reactions with two or moresubstrates

ATP + glucose → ADP +glucose 6-phosphate

The rates of bisubstrate reactions can also be analyzed by theMichaelis-Menten approach.

There are different possible mechanisms: Mech

How to distinguish between these possibilities?Steady-state kinetics can often help View

Not all enzymes fit the Michaelis-Menten model.

• Some enzymes have multiple substrates.


• Some enzyme-catalyzed reactions have many steps.


• With allosteric enzymes, binding of a substrate at one active site can affect the catalytic activity of other active sites.


• Allosteric enzymes exhibit cooperativity.• Velocity plot is sigmoidal!




4 Coenzymes


6 Catalytic RNA

Part I: Introduction to enzymesPart II: Enzyme kinetics

Inhibition of Enzyme ActivityCoenzymes

Defective enzymes and diseaseCatalytic RNA

Michaelis & Menten KineticsAllosteric enzymes kinetics

Isosteric and allosteric enzymes

One Vs Various conformations

Allosteric Enzymes: different catalytic properties, theproportion of the total number of enzyme molecules isinfluenced by substrates and other ligands

There are differences in kinetics: the affnity of allostericenzymes is not constant, but depends on the type andconcentration of the ligand. View

Allosteric enzymes

Control [regulatory] enzymes

Have quaternary structure

Have active site and modulatory site* Active site binds substrate to give product* Modulatory site binds +ve or -ve modulator toincrease or decrease the activity of the active site

Catalyse an irreversible reaction

Inhibited by end product: Feedback inhibition

Activated by substrate and other positivemodulators

Do not obey Michaelis Menten Kinetics

Allosteric Enzyme example: PFK




4 Coenzymes


6 Catalytic RNA

KEY CONCEPTS

• Noncompetitive, mixed, and uncompetitive inhibitors decrease kcat.

• Allosteric regulators can inhibit or activate enzymes.

Some inhibitors act irreversibly.

A “suicide inhibitor”

Irreversible Inhibitors

E + I → EI

E doesn’t regain activity

E-I bind in a covalent way

Suicide inhibition: occurs when an enzyme binds a substrateanalogue and forms an irreversible complex with it through acovalent bond during the “normal“ catalysis reaction.

Reversible inhibitors

E + I � EI

* E & I bind by non-covalent bonds* E I can be dissociated by: dilution, dialysis

1 Competitive

2 Uncompetitive

3 Mixed (non-competitive)

Competitive inhibition

Competitive inhibitors are generally substrate or transitionstate analogues

They have properties similar to those of one of the substrates(or transition state) of the target enzyme: competition S vs Ifor active site

Km increases but Vmax does not change

Can be reverted by high substrate concentration

Ki =[E ][I ]

[EI ]; K app

m = Km(1+[I ]

Ki)

Uncompetitive inhibition

Inhibitor binds only to the ES complex and apparently distortsthe active site

Vmax and Km decrease

Kiu =[ES ][I ]

[ESI ]

V appmax =

Vmax

(1+[I ]

Kiu); K app

m =Km

(1+[I ]

Kiu)

Vmax

Kmis unaffected

Mixed inhibition (non-competitive)

Inhibitor binds to both the enzyme and the enzymesubstratecomplex and may interfere with both substrate binding andcatalysis

The apparent Vmax decreases and the Km apparent maydecrease or increase

When only Vmax is affected, the inhibition is said to benon-competitive simple or pure

Ki =[E ][I ]

[EI ]; K

�i =

[ES ][I ]

[ESI ]

V appmax =

Vmax

(1+[I ]

Ki)

Kinetics of Inhibition

Enzymatic Inhibition: Summary

Use of enzyme inhibitors

As drugs (in pharmacology)Antiviral, Antibacterial, Anti tumor drugs

Agricultural pesticides and insecticides

Study of enzyme mechanisms of action

Diagnoses of diseases.In prostate cancer acid phosphatase is increased, L-tartrateinhibits competitively 95% of the acid phosphate fromprostrate, but has lower inhibitory effect on acid phosphatasefrom other sources. Samples from suspected carcinomapatients can be assessed in presence and absence of L-tartrate.

Example: Sulfa drugs

Example: Methotrexate

Structural analog of folic acid competes with dihydro-folatereductase.It is used for treatment of leukaemia.

Commonly Used Drugs that are Enzyme Inhibitors




4 Coenzymes


6 Catalytic RNA




Regulation of enzyme activity

Mechanisms of regulation

Temperature, pH, cofactors, coenzymes, range of substratesand location all help regulate enzymes.

pH effect

Examples of optimum pH

Enzyme Source Optimum pH

pepsin gastric mucosa 1.5sucrase intestine 6.2catalase liver 7.3arginase beef liver 9.0alkaline phosphatase bone 9.5

Temperature effect




Regulation of enzyme activity

Mechanisms of regulation

Several other methods are available to the cell for regulation:

Long term (hours or more): protein synthesisShort term (seconds): changes in catalytic efficiency

* Feed back inhibition: a kind of allosteric regulation* Regulatory covalent modifications: reversible or irreversible

Feed back inhibition

Irreversible modifications: proteolysis

Certain enzymes are synthesized and secreted as inactiveprecursor proteins known as proenzymes or zymogens

Proenzymes facilitate rapid mobilization of an activity inresponse to physiologic demand

Ex: digestive enzymes pepsin, trypsin, and chymotrypsin(proenzymes = pepsinogen, trypsinogen, andchymotrypsinogen, respectively) Prot

Reversible covalent modifications

Phosphorylation-dephosphorylation: Protein Kinases andphospatases

Involves several proteins and ATP and is under direct neuraland hormonal contro




4 Coenzymes


6 Catalytic RNA

Coenzymes

In many ez-catalyzed reactions, electrons or groups of atoms aretransferred from one substrate to another. This type of reactionalways also involves additional molecules (coenzymes), whichtemporarily accept the group being transferred.

Coenzymes can be soluble (S) or act as a prostetic group (P).

Coez are chemically changed by the enzymatic reactions in whichthey participate: in order to complete the catalytic cycle, thecoenzyme must be returned to its original state.

For prosthetic groups, this can occur only in a separate phase of theenzymatic reaction sequence. For transiently bound coenzymes,such as NAD, the regeneration reaction may be catalyzed by adifferent enzyme.

Redox coenzymes and Group-transferring coenzymes




4 Coenzymes


6 Catalytic RNA

Some examples

A number of hereditary diseases result from the absence of anenzyme or a defective one.




Phenylketonuria (PKU)

Genetic defect that results in a defect of the enzymephenylalanine hydroxylase.

Affects about 1 baby per 13,000.

Children are screened at birth.

Can result in retarded physical and mental development ifuntreated.

Treatment: restrict phenylalanine until age 10 (until brain isdeveloped).

PKU is one of a family of enzymatic/genetic disorders relatedto phenylalanine metabolism.

Phenylketonuria (PKU)




4 Coenzymes


6 Catalytic RNA

Catalytic RNA

Since their discovery, it was believed that all enzymes wereproteins.

In 1981 - 1982, two research group reported results oncatalytic RNA.

In 1989, the Nobel Prize in Chemistry was awarded to SidneyAltman (Yale) and Thomas Cech (University of Colorado -Boulder) for their discovery.

The term ribozyme is now used for RNA enzymes.

Ribonuclease P

This was the first type of catalytic RNA discovered and is presentin all organisms

Substrates are at least 60 inactive, precursor forms of tRNA.

Ribonuclease P acts to remove a segment of theribonucleotide, producing mature tRNA.

The enzyme consists of a small protein subunit with amolecular weight of 14,000 and an RNA component of 377nucleotides.




Significance of ribozymes

Other forms of catalytic RNA continue to be discovered.

Small ribozymes have been found as components of plantRNA viruses.

The active region of this RNA consists of only 19 - 30nucleotides.

Because of their characteristic shape and action, they arecalled “hammerhead” ribozymes.




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Proteolysis example

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Enzimas 2015 01 BqaBasica

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