ENZYMES AS CATALYSTS ROLE OF COENZYMES AND METALS

IN ENZYME CATALYSIS

Associate Professor Ana Savic Radojevic

ENZYMES There are two fundamental conditions for life:

1. the living entity must be able to self-replicate

2. the organism must be able to catalyze chemical reactions efficiently and selectively

Without catalysis, chemical reactions could not occur on a useful time scale, and thus could not sustain life.

The conversion of sucrose to CO2 and H2O in the presence of oxygen is a highly exergonic process, releasing free energy that we can use to think, move, taste, and see. However, a bag of sugar can remain on the shelf for years without any obvious conversion to CO2 and H2O. Yet when sucrose is consumed by a human, it releases its chemical energy in seconds. The difference is catalysis.

ENZYMES, THE MOST REMARKABLE AND HIGHLY SPECIALIZED PROTEINS

Enzymes have extraordinary catalytic power:

high degree of specificity for their substrates,

accelerate chemical reactions tremendously,

function in aqueous solutions under very mild conditions of temperature and pH.

Enzymes are central to every biochemical process; through the action of regulatory enzymes, metabolic pathways are highly coordinated.

The study of enzymes has immense practical importance:

In some genetic disorders, there may be a deficiency or even a total absence of one or more enzymes.

Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are important in diagnosing certain illnesses.

Many drugs exert their biological effects through interactions with enzymes.

Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach.

In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by “ferments” .

The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early enzyme studies. Sumner found that urease crystals consisted entirely of protein, and he postulated that all enzymes are proteins.

Haldane made the remarkable suggestion that weak bonding interactions between an enzyme and its substrate might be used to catalyze a reaction.

Much of the history of biochemistry is the history of enzyme research

ENZYMES ARE CLASSIFIED BY REACTION TYPE: INTERNATIONAL CLASSIFICATION OF ENZYMES

this system divides enzymes into six classes, each with subclasses, based on the type of reaction catalyzed

Enzyme names

Most enzyme names end in “ase.” Enzymes usually have both:

o a common name and

o a systematic classification that includes a name and an Enzyme Commission (EC) number.

Each enzyme is assigned a four-part classification number, Enzyme Commission number (E.C. number). The formal systematic name of the enzyme catalyzing the reaction:

is ATP:glucose phosphotransferase, which indicates that it catalyzes the transfer of a phosphoryl group from ATP to glucose. E.C. number is 2.7.1.1.

(2) denotes the class name (transferase); (7) the subclass (phosphotransferase); (1) a phosphotransferase with a hydroxyl group as acceptor; (1) D-glucose as the phosphoryl group acceptor.

For many enzymes, a trivial name is more commonly used—in this case hexokinase.

How enzymes work

Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 105 to 1017.

Enzyme-catalyzed reactions are characterized by the formation of a complex between substrate and enzyme (an ES complex).

Substrate binding occurs in a pocket on the enzyme called the ACTIVE SITE.

The function of enzymes is to lower the activation energy, G‡ thereby enhance the reaction rate. The equilibrium of a reaction is unaffected by the enzyme.

Enzyme binding sites: • Active site • Allosteric site in allosteric enzymes

Specificity of enzyme: active site The active site is usually a cleft or crevice in the enzyme formed by one or more regions of the polypeptide chain. Initially, the substrate molecules bind to their substrate binding sites, also called the substrate recognition sites. The three-dimensional arrangement of binding sites in a crevice of the enzyme allows the reacting portions of the substrates to approach each other from the appropriate angles.

The active site : • Substrate binding site • Active catalytic site

The substrate binding site overlap in the active catalytic site of the enzyme, the region of the enzyme where the reaction occurs.

THE ACTIVE SITE OF THE ENZYME

Enzyme specificity (the enzyme’s ability to react with just one substrate) results from the three-dimensional arrangement of specific amino acid residues in the enzyme that form binding sites for the substrates and activate the substrates during the course of the reaction.

INDUCED FIT, a mechanism postulated by Daniel Koshland in 1958 :

1. Induced fit serves to bring specific functional groups on the enzyme

into the proper position to catalyze the reaction.

2. The conformational change also permits formation of additional weak

bonding interactions in the transition state.

In either case, the new enzyme conformation has enhanced catalytic

properties.

Induced fit is a common feature of the reversible binding of ligands to

proteins. Induced fit is also important in the interaction of almost every

enzyme with its substrate.

” Induced fit” model for substrate binding

As the substrate binds, enzymes undergo a

conformational change (“induced fit”) that repositions

the side chains of the amino acids in the active site and

increases the number of binding interactions.

The substrate binding site is not a rigid “lock” but rather

a dynamic surface created by the flexible overall three-

dimensional structure of the enzyme. Conformational change resulting from the binding of glucose to hexokinase.

MECHANISM OF CATALYSIS

Catalysts increase the rate of chemical reactions by decreasing the activation energy (Ea)

Catalysts do not change the concentration of substrates and products at equilibrium, but they do allow equilibrium to be reached more rapidly

No permanent change in catalysts occurs during the reactions they catalyze

Enzymes are biologycal catalysts . A simple enzymatic reaction might be written

E + S ↔ ES ↔ EP ↔ E + P

Enzymes affect reaction rates, not equilibria

THE GROUND STATE (S OR P):The starting point for either the forward or the reverse reaction. TRANSITION STATE: the point at the top of the energy hill at which decay to the S or P state is equally probable

The transition state is not a chemical species with any significant stability and should not be confused with a reaction intermediate (such as ES or EP). It is a fleeting molecular moment in which events such as bond breakage/formation, and charge development have proceeded to the precise point at which decay to either substrate or product is equally likely.

The difference between the energy levels of the ground state and the transition state is the ACTIVATION ENERGY, ΔG‡.

In the coordinate diagram, the free energy of the system is plotted against the progress of the reaction (the reaction coordinate).

“STICKASE”

Chemical reactions of many types take place between substrates and enzymes’ functional groups (specific amino acid side chains, metal ions, and coenzymes).

The interaction between substrate and enzyme in this complex is mediated by the same forces that stabilize protein structure, including hydrogen bonds and hydrophobic and ionic interactions

Much of the energy required to lower activation energies is derived from weak, noncovalent interactions between substrate and enzyme.

What really sets enzymes apart from most other catalysts is the formation of a specific ES complex.

Activation energy and the transition state

The functional groups in the catalytic site of the enzyme activate the substrates and decrease the energy needed to form the high-energy intermediate stage of the reaction known as the transition state complex.

Some of the catalytic strategies employed by enzymes are general acid-base catalysis, formation of covalent intermediates, and stabilization of the transition state.

A few principles explain the catalytic power and specificity of enzymes

A. The rearrangements of covalent bonds during an enzyme-catalyzed reaction • Catalytic functional groups on an enzyme may form a transient covalent bond with a

substrate. • These interactions lower the activation energy by providing an alternative, lower-

energy reaction path. B. The noncovalent interactions between enzyme and substrate Formation of each weak interaction in the ES complex is accompanied by release of a small amount of free energy that provides a degree of stability to the interaction. This energy is called binding energy, GB. Binding energy is a major source of free energy used by enzymes to lower the activation energies of reactions:

1. This binding energy contributes to specificity as well as to catalysis. 2. Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrates per se but to the transition states through which substrates pass as they are converted to products during an enzymatic reaction.

MECHANISM OF CATALYSIS IN ENZYME REACTIONS

General acid-base catalysis- Many reactions involve the formation of unstable charge intermediates that tend to break down rapidly to their constituent reactant. They can be often stabilized by the transfer of protons to or from the substrate or intermediate to form a species that breaks down more rapidly to products than to reactants

Covalent catalysis- In this type of catalysis, a transient covalent bond is formed between the enzyme and the substrate

Metal ion catalysis-Metals can participate in catalysis in several ways. Ionic interactions between an enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states. They can also mediate oxidation-reduction reactions by reversible change in the metal,ions oxidation state

A - B + X: → A - X + B → A + X: + B

Functional groups on amino acid side chains in the active site

MECHANISM OF GENERAL AND SPECIFIC ACID-BASE CATALYSIS

Without catalysis, unstable (charged) intermediate breaks down rapidly to form reactants

Reactants

Proton donor and acceptor is molecule of water

Proton donor and acceptor are any acid (HA) and any base (B:)

Products

Amino acids in general acid-base catalysis

LYSOZYME: A COMBINATION OF ENZYME-INDUCED SUBSTRATE STRAIN AND ACID-BASE CATALYSIS

Heksasaharid-supstrat

Mesto

hidrolize

Heksasaharid-supstrat

Mesto

hidrolize

Konformacija

polustolice

Konformacija

stolice

Konformacija

polustolice

Konformacija

stolice

Conformation of a suggar residue “D” at which bond breaking occurs is strained from the stable chair to the unstable half-chair conformation upon binding The concept of substrate strain explains the role of the enzyme in increasing the rate of reaction

Hexasaccharide substrate

Site of hydrolysis

Half-chair conformation

Chair conformation

Mechanism for lysozyme action I

Glutamate 35 acts as a proton donor to the glycosidic bond

Glycosidic bond is cleaved and a carbonium ion is formed (positive charge on carbon 1)

disaccharide NAG leaves enzyme molecule (product 1)

proizvod

-

C

O

O

Glu 35

O

C1

NAG3

O

C4

O

D

E

-

C

O

O

Glu 35

H

C

O

O

Asp 52

-

H

NAG3

O

C4

O

D

E

NAG

H

O

C1

+

C

O

O

Asp 52

-

NAG

P1

Product P1

Mechanism for lysozyme action II

carbonium ion intermediate reacts with OH-group from water

tetra-NAG (product 2) leaves enzyme molecule

glutamate 35 is reprotonated with H+ from water and enzyme is ready for new catalysis

-

C

O

O

Glu 35

NAG3

D

H

O

C1

+

C

O

O

Asp 52

-

HO

H

C

O

O

Asp 52

-

C

O

O

Glu 35

H

NAG3

D

H

O

C1

HO

P2

proizvodProduct P2

CATALYTIC MECHANISM OF CHYMOTRYPSIN

• Chymotrypsin is a digestive enzyme that catalyzes the hydrolysis of specific peptide bonds in denatured proteins. It is a member of the serine protease superfamily, enzymes that use a serine in the active site to form a covalent intermediate during proteolysis.

•In the overall hydrolysis reaction the carbonyl carbon, which carries a partial positive charge, is attacked by a hydroxyl group from water. An unstable tetrahedral oxyanion intermediate is formed, which is the transition state complex. As the electrons return to the carbonyl carbon, it becomes a carboxylic acid, and the remaining proton from water adds to the leaving group to form an amine

1. Substrate binding

2. Histidine activates serine for nucleophilic attack

I stage:cleavage of the peptide bond in the denatured substrate and formation of a covalent acyl-enzyme intermediate

As the substrate protein binds to the active site, Ser195 and His57 are moved closer together for the nitrogen electrons on His to attract the hydrogen of Ser. Without this change of conformation on substrate binding, the catalytic triad cannot form.

His serves as a general base catalyst as it abstracts a proton from the Ser, increasing the nucleophilicity of the serine-oxygen, which attacks the carbonyl carbon.

3. The oxyanion tetrahedral intermediate is stabilized by hydrogen bonds

4. cleavage of the peptide bond

I stage:cleavage of the peptide bond in the denatured substrate and formation of a covalent acyl-enzyme intermediate

The electrons of the carbonyl group form the oxyanion tetrahedral intermediate. The oxyanion is stabilized by the NH groups of Ser195 and glycine

The amide nitrogen in the peptide bond is stabilized by interaction with the His proton (general acid catalysis). As the electrons of the peptide bond withdraw into the nitrogen, the electrons of the carboxyanion return to the substrate carbonyl carbon, resulting in cleavage of the peptide bond.

5. The covalent acyl–enzyme intermediate

I stage: cleavage of the peptide bond in the denatured substrate and formation of a covalent acyl-enzyme intermediate

The cleavage of the peptide bond results in formation of the covalent acyl-enzyme intermediate, and the amide half of the cleaved protein dissociates

6. Water attacks the carbonyl carbon

7. Second oxyanion tetrahedral intermediate

STAGE II - hydrolysis of the acyl-enzyme intermediate

The active site His activates water to form an OH- for a nucleophilic attack.

Second oxyanion transition state is formed. The oxyanion is again stabilized by the NH groups of Ser195 and glycine in the chymotrypsin peptide backbone

8. Acid catalysis breaks the acyl–enzyme covalent bond

9. The product is free to dissociate

STAGE II - hydrolysis of the acyl-enzyme intermediate

MMMMMMMM

The active site histidine adds the proton back to serine .

The reaction is complete and the product dissociates .

Energy diagram in the presence of chymotrypsin

Mechanism-based inhibitors.

The effectiveness of many drugs and toxins depends on their ability to inhibit an enzyme.

The strongest inhibitors are covalent inhibitors, compounds that form covalent bonds with a reactive group in the enzyme active site, or transition state analogues that mimic the transition state complex.

CATALYTIC RESIDUES ARE HIGHLY CONSERVED

Members of an enzyme family such as the aspartic or serine proteases employ a similar mechanism to catalyze a common reaction type but act on different substrates. Most enzyme families arose through gene duplication events that create a second copy of the gene that encodes a particular enzyme.

The proteins encoded by the two genes can then evolve independently to recognize different substrates—resulting, for example, in chymotrypsin, which cleaves peptide bonds on the carboxyl terminal side of large hydrophobic amino acids, and trypsin, which cleaves peptide bonds on the carboxyl terminal side of basic amino acids.

Proteins that diverged from a common ancestor are said to be homologous to one another.

The common ancestry of enzymes can be inferred from the presence of specific amino acids in the same position in each family member. These residues are said to be conserved residues.

Among the most highly conserved residues are those that participate directly in catalysis.

ISOZYMES ARE DISTINCT ENZYME FORMS THAT CATALYZE THE SAME REACTION Higher organisms often elaborate several physically distinct versions of a given enzyme, each

of which catalyzes the same reaction. Like the members of other protein families, these protein catalysts or isozymes arise through gene duplication. Isozymes may exhibit subtle differences in properties such as sensitivity to particular regulatory factors or substrate affinity (eg, hexokinase and glucokinase) that adapt them to specific tissues or circumstances. Some isozymes may also enhance survival by providing a “backup” copy of an essential enzyme.

Prosthetic groups, cofactors and coenzymes

• Prosthetic groups, cofactors and coenzymes – are small nonprotein molecules and metal ions that participate directly in substrate binding or catalysis

• Holoenzyme is catalyticly active enzyme with it’s coenzyme or metal ion, protein part of holoenzyme is apoenzyme

Prosthetic Groups Are Tightly Integrated Into an Enzyme’s Structure

Prosthetic groups are distinguished by their tight, stable incorporation into a protein’s structure by covalent or noncovalent forces. Examples include pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn, and Zn.

Metals are the most common prosthetic groups. The roughly one-third of all enzymes that contain tightly bound metal ions are termed metalloenzymes. Metal ions that participate in redox reactions generally are complexed to prosthetic groups such as heme or iron-sulfur clusters.

Metals also may facilitate the binding and orientation of substrates, the formation of covalent bonds with reaction intermediates (Co2+ in coenzyme B12), or interact with substrates to render them more electrophilic (electron-poor) or nucleophilic (electron-rich).

Cofactors Associate Reversibly With Enzymes or Substrates

Cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably associated prosthetic groups, cofactors therefore must be present in the medium surrounding the enzyme for catalysis to occur.

The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which metal ions serve as prosthetic groups.

Coenzymes Serve as Substrate Shuttles

Coenzymes serve as recyclable shuttles—or group transfer agents—that transport many substrates from their point of generation to their point of utilization. Association with the

coenzyme also stabilizes substrates such as hydrogen atoms or hydride ions that are unstable in the aqueous environment of the cell. Other chemical moieties transported by coenzymes

include methyl groups (folates), acyl groups (coenzyme A), and oligosaccharides (dolichol).

Many Coenzymes, Cofactors & Prosthetic Groups Are Derivatives of B Vitamins

The water-soluble B vitamins supply important components of numerous coenzymes. Several coenzymes contain, in addition, the adenine, ribose, and phosphoryl moieties of AMP or ADP. Nicotinamide is a component of the redox coenzymes NAD and NADP, whereas riboflavin is a component of the redox coenzymes FMN and FAD.

Pantothenic acid is a component of the acyl group carrier coenzyme A.

As its pyrophosphate, thiamin participates in decarboxylation of α-keto acids, and folic acid and cobamide coenzymes function in one-carbon metabolism.

Some inorganic elements that serve as cofactors for enzymes

Cu2+ cytohrome oxidase

Fe2+ ili Fe3+ cytohrome oxidase, catalase, peroxydase

K+ pyruvate kinase

Mg2+ hexokinase, glucoso 6-phosphatase, pyruvate kinase

Mn2+ ribonucleotide reductase

Se glutathione peroxydase

Zn2+ carbonic anhydrase, carboxypeptidase A & B

Some coenzymes that serve as transient carries of specific atoms or functional groups

Coenzyme Chemical group for transfer Vitamin

Biotin CO2 biotin

Coenzyme A (CoA) acyl group pantothenic acid

Coenzyme B12 (CoB12) H atom / alkyl group cobalamine(B12)

Flavin adenine dinucleotide (FAD)

electrons riboflavin (B2)

Nicotinamide adenine dinucleotide (NAD)

hydride ion (H-) niacine

Pyridoxal phosphate amino group pyridoxine (B6)

Tetrahydrofolate one-carbon fragments folic acid

Thiamine diphosphate aldehyde thiamine (B1)

Role of metals in catalysis

Metalloenzymes are enzymes that contain tightly bound metal ions

Sometimes metal ions are part of prosthetic nonprotein group such as heme

Enzymes that require a metal ion cofactor are termed metal-activated

enzymes. In that case metal isn’t integrated into an enzyme’s structure

Role of metals: A. stabilize active conformation of enzyme; B. also may facilitate the binding and orientation of substrate; C. can act as direct catalysts; D. have an important role in oxido-reduction reactions

Metals cofactors have various functions

• Some transition metals like Zn, Fe, Mn i Cu can act as Lewis acid, because they have empty d electron orbitals

• A good example of a metal functioning as a Lewis acid is found in carbonic anhydrase (zinc enzyme). The first step is generation of a proton and a hydroxyl group which binds to the zinc. The proton and hydroxyl group are then added to the carbon dioxide and carbon acid is relaesed

Role of the metals Enz-S-M: “Substrate-bridged” complex

1. The true substrate for creatine kinase is not ATP, but Mg2+-ATP. In this case Mg2+ does not interact directly with the enzyme, but may serve to neutralize the negative charge density on ATP and faciliate binding to the enzyme

2. Scheme for the binding of Mg2+-ATP and glucose in the active site of hexokinase

3. All kinases exept muscule pyruvate kinase and phosphoenolpyruvate kinase are“Substrate-bridged” complexes

Enz-M-S: “Metal-bridged” complex

• This enzymes contain a tightly bound transitional metals (Zn ili Fe)

Model of the role of K+ in the active site of pyruvate kinase

A. It belongs to Enz-M-S bridged

complexes. Mg coordinates the substrate to the enzyme active site

B. Initial binding of K+ induces conformational changes in the kinase, which results in increased affinity for phosphoenol-pyruvate. In addition K+ orients the phosphoenol-pyruvate in the correct position for transfer of it’s phosphate to ADP, the second substrate

Thus, K+ and Na+, stabilize active

conformation of the enzyme but are passive in cataysis.

phosphoenol-pyruvate +ADP ATP + pyruvate pyruvate kinase

pH and temperature profiles.

Enzymes have a functional pH range determined by the pKa of functional groups in the active site and the interactions required for three-dimensional structure.

Non-denaturing increases of temperature increase the reaction rate.

Catalytic RNAs: RIBOZYMES

Some of the most interesting molecular events in RNA metabolism occur during this postsynthetic processing. Intriguingly, several of the enzymes that catalyze these reactions consist of RNA rather than protein.

The discovery of these catalytic RNAs, or ribozymes, has brought a revolution in thinking about RNA function and about the origin of life.

RNA Catalyzes the Splicing of Introns

Self-splicing group I introns share several properties with enzymes besides accelerating the reaction rate, including their kinetic behaviors and their specificity. Because the intron itself is chemically altered during the splicing reaction—its ends are cleaved—it may appear to lack one key enzymatic property: the ability to catalyze multiple reactions. Most of the activities of these ribozymes are based on two fundamental reactions: transesterification and phosphodiester bond hydrolysis (cleavage). The substrate for ribozymes is often an RNA molecule, and it may even be part of the ribozyme itself.

The enzymatic activity that catalyzes peptide bond formation has historically been referred to as peptidyl transferase and was widely assumed to be intrinsic to one or more of the proteins in the large ribosomal subunit.

We now know that this reaction is catalyzed by the 23S rRNA, adding to the known catalytic repertoire of ribozymes. This discovery has interesting implications for the evolution of life

Enzyme-Linked Immunoassays

The sensitivity of enzyme assays can be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunosorbent assays (ELISAs) use antibodies covalently linked to a “reporter enzyme” such as alkaline phosphatase or horseradish peroxidase whose products are readily detected, generally by the absorbance of light or by fluorescence.

Serum or other biologic samples to be tested are placed in a plastic microtiter plate, where the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then “blocked” by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and are themselves immobilized.

Excess free antibody molecules are then removed by washing.

The presence and quantity of bound antibody is then determined by adding the substrate for the reporter enzyme.

Additional catalytic mechanisms include GENERAL ACID-BASE CATALYSIS, COVALENT CATALYSIS, AND METAL ION CATALYSIS. Catalysis often involves transient covalent interactions between the substrate and the enzyme, or group transfers to and from the enzyme to provide a new, lower-energy

reaction path

The binding energy, GB, can be used to lower substrate entropy or to cause a conformational change in the enzyme (INDUCED FIT). Binding energy also accounts for the exquisite specificity of enzymes for their

substrates.

Enzyme-catalyzed reactions are characterized by the formation of an ES complex. Substrate binding occurs in a pocket on the enzyme called

the active site

Some of these weak interactions occur preferentially in the reaction transition state, thus stabilizing the transition state.

Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 105 to 1017.

A significant part of the energy used for enzymatic rate enhancements is derived from WEAK INTERACTIONS (hydrogen bonds and hydrophobic and ionic

interactions) between substrate and enzyme.