Enzymatic catalysis

Chapter 11

Enzymatic catalysisRole of enzymes Role of enzymes serve the same role as any other catalyst in serve the same role as any other catalyst in

chemistrychemistry

act with a higher specificityact with a higher specificity

acid and base catalyst possible due to proximal acid and base catalyst possible due to proximal locations of amino acidslocations of amino acids

alter the structure location of quantity of the alter the structure location of quantity of the enzyme and you have regulated the formation of enzyme and you have regulated the formation of product - try that in organic chemistry !product - try that in organic chemistry !

Don't lose sight of what enzymes do. Straight Don't lose sight of what enzymes do. Straight forward reactions…. reactants -> products.forward reactions…. reactants -> products.

Naming enzymes - Naming enzymes - Enzyme commission for each enzyme based on the Enzyme commission for each enzyme based on the

type of reactions. Kind of like IUPACtype of reactions. Kind of like IUPAC Yeah right - the mother of invention – he/she who Yeah right - the mother of invention – he/she who

finds it names if. Trivial names rulefinds it names if. Trivial names rule

Enzyme reaction types: Enzymes are Enzyme reaction types: Enzymes are classified into six major classes based on classified into six major classes based on the reaction they catalyze.the reaction they catalyze.

1) 1) OxidoreductasesOxidoreductases catalyze oxidation-reduction catalyze oxidation-reduction reactions. Most of these enzymes are known as reactions. Most of these enzymes are known as dehydrogenases, but some are called oxidases, dehydrogenases, but some are called oxidases, peroxidases, oxygenases or reductases.peroxidases, oxygenases or reductases.

A- + B -> A + B-

A is the reductant (e- donar) and B is the oxidant (e- acceptor)

NAD+ (ox) +e- + H+ -> NADH (red)catalyzes the transfer of electrons from one molecule (the reductant, also called the hydrogen or electron donor) to another

(the oxidant, also called the hydrogen or electron acceptor).

2) 2) TransferasesTransferases catalyze group-transfer catalyze group-transfer reactions (functional groups). Many require reactions (functional groups). Many require the presence of coenzymes. A portion of the the presence of coenzymes. A portion of the substrate molecule usually binds covalently to substrate molecule usually binds covalently to these enzymes or their coenzymes. This these enzymes or their coenzymes. This group includes my personal favorite, the group includes my personal favorite, the kinaseskinases

A-X + B -> A + B-XA is the donar and B is the acceptor Donor s often a coenzyme

ATP + R-OH -> ADP + R-PO4-2

3) 3) HydrolasesHydrolases catalyze hydrolysis. They are a catalyze hydrolysis. They are a special class of transferases, with water serving special class of transferases, with water serving as the acceptor of the group transferredas the acceptor of the group transferred

A-B + H2O -> A-OH + B-H

ie Glycoside hydrolases (also called glycosidases) catalyze the hydrolysis of the glycosidic linkage to generate two smaller sugars.

They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose and hemicellulose

Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.

4)4)LyasesLyases catalyze nonhydrolytic and catalyze nonhydrolytic and nonoxidative elimination reactions, or lysis, of nonoxidative elimination reactions, or lysis, of a substrate, generating a double bond. In a substrate, generating a double bond. In other words these reactions catalyze group other words these reactions catalyze group elimination to form double bonds. In the elimination to form double bonds. In the reverse direction, lyases catalyze addition of reverse direction, lyases catalyze addition of one substrate to a double bond of a second one substrate to a double bond of a second substrate. substrate.

A lyase that catalyzes addition reaction in cells A lyase that catalyzes addition reaction in cells is often termed a synthase.is often termed a synthase.

Usually only need one substrate in one direction Usually only need one substrate in one direction and two for the reverse!and two for the reverse!

ATP -> cAMP + PPi

5) 5) IsomerasesIsomerases catalyze the isomerase reactions. catalyze the isomerase reactions.

catalyses the structural rearrangement of catalyses the structural rearrangement of isomersisomers

Because these reactions have only one Because these reactions have only one substrate and one product, they are among substrate and one product, they are among the simplest enzymatic reactions.the simplest enzymatic reactions.

A -> B

Names of these enzymes are “substrate isomersase”Ex. - enoyl CoA isomerase catalyzes conversion of cis-double bonds of fatty acids at position 3 to trans double bonds at position 2. It has a special importance in metabolism of unsaturated fatty acids

or some sort of mutase to form of carbohydrate

6) 6) LigasesLigases catalyze ligation or joining, of two catalyze ligation or joining, of two substrates. These reactions require the input substrates. These reactions require the input of chemical potential energy of a nucleoside of chemical potential energy of a nucleoside triphosphate such as ATP. i.e. bond formation triphosphate such as ATP. i.e. bond formation coupled to ATP hydrolysis. Ligases are usually coupled to ATP hydrolysis. Ligases are usually referred to as synthetases.referred to as synthetases.

Ab + C -> A-C + b

Sometimes involve a hydrolysisA and C are both large polymers DNA ligase

General Information on General Information on EnzymologyEnzymology

Enzyme nomenclature Enzyme nomenclature • Active siteActive site• Substrate vs. reactantSubstrate vs. reactant• Prosthetic groups, coenzyme and cofactorsProsthetic groups, coenzyme and cofactors• Activity vs. reactionActivity vs. reaction• Suffix -aseSuffix -ase

Another important reminder - not all activity is on or Another important reminder - not all activity is on or off. Many times the enzyme has a low or off. Many times the enzyme has a low or constitutive activity that can be increased many constitutive activity that can be increased many times. times. It is a common mistake to think of enzymes It is a common mistake to think of enzymes being on or offbeing on or off..

Enzyme specificityEnzyme specificity

- One of the most powerful actions of enzymesOne of the most powerful actions of enzymes. . -

– Group complementationGroup complementation - the ability to - the ability to recognize specific regions of the substrate to recognize specific regions of the substrate to align reactants with catalytic site.align reactants with catalytic site.

– Based on non-covalent molecular interactions.Based on non-covalent molecular interactions.

– Lock and key vs. induced fit - both occur. Lock and key vs. induced fit - both occur. Induced fit takes place when binding of one part Induced fit takes place when binding of one part of the substrate to the enzyme alters the of the substrate to the enzyme alters the conformation of the enzyme to make a true "fit"conformation of the enzyme to make a true "fit"

– Binding does not mean activityBinding does not mean activity

Enzyme specificityEnzyme specificity

An enzyme can bind and react stereo-specifically with An enzyme can bind and react stereo-specifically with chiral compoundschiral compounds

• This can happen due to a three point attachmentThis can happen due to a three point attachment– Binding can then only occur in one way and Binding can then only occur in one way and

therefore the products are not a mixture.therefore the products are not a mixture.

3 pt attachment demonstrating the sterio-specificityon an enzyme binding to its substrate

Enzyme binding site

Substrate

How do enzymes work?How do enzymes work?

• By reducing the energy of activationBy reducing the energy of activation• This happens because the transition state is stabilized by a number of This happens because the transition state is stabilized by a number of

mechanisms involving the enzyme/proteinmechanisms involving the enzyme/protein• Without enzymes reactions occur by collisions between reactants or Without enzymes reactions occur by collisions between reactants or

addition of various organic catalystsaddition of various organic catalysts• The energy barrier between the reactants and product, is the free The energy barrier between the reactants and product, is the free

energy of activation.energy of activation.• The free energy (The free energy (G) of G) of

the reaction is what the reaction is what

determines if a reactiondetermines if a reaction

is spontaneous.is spontaneous.

How do enzymes work?How do enzymes work? • The rate is determined by That's why an enzyme can increase The rate is determined by That's why an enzyme can increase

the speed / rate of a reaction.the speed / rate of a reaction.• The overall free energy of a reaction, The overall free energy of a reaction, G, is independent of the G, is independent of the

free energy of activation. So if the free energy of activation. So if the G is less than zero the G is less than zero the reaction will proceed once a catalyst is added (the enzyme). reaction will proceed once a catalyst is added (the enzyme). – But the converse is also true - the reverse reaction is also But the converse is also true - the reverse reaction is also

able to proceed depending on the able to proceed depending on the G. Some reactions have G. Some reactions have very littlevery little

changes free changes free energy and are energy and are freely reversible. freely reversible. Availability of Availability of enzyme (catalyst) enzyme (catalyst) and the relative and the relative concentrations of concentrations of products and products and reactants will reactants will decide which decide which direction these direction these reactions will go.reactions will go.

Factors effecting catalysisFactors effecting catalysis

Temperature –Temperature – Unlike conventional chemical Unlike conventional chemical reactions, increasing temperature of a reaction can reactions, increasing temperature of a reaction can have mixed results.have mixed results. Initial increases in temperature result in higher Initial increases in temperature result in higher

rates of reaction due to increasing the kinetic rates of reaction due to increasing the kinetic activity of reacting molecule – more forceful activity of reacting molecule – more forceful collisions and higher rates of diffusion between collisions and higher rates of diffusion between substrate and its enzymesubstrate and its enzyme

Increased temp exceeds energy barrier to Increased temp exceeds energy barrier to maintain hydrogen and hydrophobic bonds of the maintain hydrogen and hydrophobic bonds of the enzyme. This results in denaturation of and loss enzyme. This results in denaturation of and loss of enzyme functionof enzyme function

Factors effecting catalysisFactors effecting catalysis

pH –pH – There is typically an optimum pH of a reaction for There is typically an optimum pH of a reaction for both substrate and enzyme. The aa in the catalytic both substrate and enzyme. The aa in the catalytic site as well as for structure is vital for the reaction site as well as for structure is vital for the reaction to occur. Most enzymes are stable in the pH range to occur. Most enzymes are stable in the pH range of 5 – 9.of 5 – 9.

Example - Example - EnzEnz-- + SH + SH++ -> EnzSH -> EnzSH

at low pHat low pH Enz - -> EnzH Enz - -> EnzH

at high pHat high pH SH -> S- + H+ SH -> S- + H+

General catalytic mechanisms of General catalytic mechanisms of enzyme reactionsenzyme reactions

- also known as the exact means that enzymes lower the - also known as the exact means that enzymes lower the transition state and catalyze the reaction.transition state and catalyze the reaction.

• There are six general mechanisms by which enzymes There are six general mechanisms by which enzymes act. These are the contributing factors of enzyme act. These are the contributing factors of enzyme catalysis:catalysis:

Remember allosteric regulations?

• Allosteric interactionAllosteric interaction – – of, relating to, of, relating to, undergoing, or being a change in the shape undergoing, or being a change in the shape and activity of a protein (as an enzyme) that and activity of a protein (as an enzyme) that results from combination with another results from combination with another substance at a point other than the chemically substance at a point other than the chemically active siteactive site

• a regulatory mechanism where a a regulatory mechanism where a small molecule (effector) binds small molecule (effector) binds and alters an enzymes activityand alters an enzymes activity

Allosteric interactionAllosteric interaction• Each allosteric unit, called

a protomer (generally assumed to be a subunit), can exist in two different conformational states - designated 'R' (for relaxed) or 'T' (for tense) states.

• The R state has a higher affinity for the ligand than the T state.

• Because of that, although the ligand may bind to the subunit when it is in either state, the binding of a ligand will increase the equilibrium in favor of the R state.

Allosteric interactionAllosteric interaction• Allosteric sites are sites on the enzyme

that bind to molecules in the cellular environment.

• The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme.

• This change in conformation translates to the active site, which then affects the reaction rate of the enzyme.

•  Allosteric interactions can both inhibit and activate enzymes and are a common way that enzymes are controlled in the body.

Acid-Base Catalysis Chemical groups can often be made more reactive by adding or removing a proton.

Enzyme active sites contain side chain groups that act as proton donors or acceptors.

These groups are referred to as general acids or general bases. The amino acid used in the reaction depends on the pH such that the side chain is in the appropriate charge state, and the local environment.

Histadine is a good example of this. The side chain of histidine has a pKa of around 6. Therefore the side chain can undergo ionization at a physiological pH.

The protonated form is a general acid and ionized form is the general base. The task of a catalyst is often to make a reactive group more reactive by increasing its intrinsic electrophilic or neutrophilic character.

The easiest way to do this is to remove or add a proton. It is not practical to undergo this mechanism by free acids or bases and at physiological pH, the concentration of OH- and H+ is too low to induce the catalysis.

Therefore the correct placement of an amino acid that can act as a base or acid effectively increases the concentration at the correct location of the substrate.

See box 11-1 for pH effects on catalysis (pp 323) RNase A is an example of this type of reaction

Covalent CatalysisCovalent Catalysis

In some enzymes a nucleophilic side chain group In some enzymes a nucleophilic side chain group forms an unstable covalent bond with the forms an unstable covalent bond with the substrate. The enzyme-substrate complex them substrate. The enzyme-substrate complex them forms product. The pathway can require that forms product. The pathway can require that the intermediate is more susceptible to the intermediate is more susceptible to nucleophilic attack by water than the original nucleophilic attack by water than the original substrate.substrate.

• Three stages of covalent catalysis:Three stages of covalent catalysis: Formation of a bond between substrate and Formation of a bond between substrate and

enzymeenzyme Removal of electrons to make a reactive Removal of electrons to make a reactive

centercenter Elimination of the bond that was formed in Elimination of the bond that was formed in

step onestep one

Metal Ion CatalysisMetal Ion Catalysis Transition metals as well as Transition metals as well as divalent cations (Mg+2 and Ca+2) are useful for divalent cations (Mg+2 and Ca+2) are useful for this type of catalysis. Metal ions act as a lewis this type of catalysis. Metal ions act as a lewis acid and accept electrons. Therefore they are acid and accept electrons. Therefore they are effective electrophiles. Another important reason effective electrophiles. Another important reason for involving metals is the positive charge at any for involving metals is the positive charge at any physiological pH.physiological pH.

Involved in redox reactionsInvolved in redox reactions Metals such as zinc activate water - to Metals such as zinc activate water - to

"acidify" or polarize the water so the OH "acidify" or polarize the water so the OH group can act as a nucleophile.group can act as a nucleophile.

Carbonic anhydrase is an example of this Carbonic anhydrase is an example of this type of reactiontype of reaction

ZnZn2+2+ in carbonic anhydrase – Zn in carbonic anhydrase – Zn2+2+ is coordinated by 3 imidazole groups is coordinated by 3 imidazole groups and a water molecule.and a water molecule.

(Arrow shows opening of active site)(Arrow shows opening of active site)

Proximity and Strain (Orientation) EffectsProximity and Strain (Orientation) Effects For a biochemical For a biochemical reaction to occur, the substrate most come into close reaction to occur, the substrate most come into close proximity to catalytic functional groups (side chains of proximity to catalytic functional groups (side chains of amino acids which are involved in catalytic reactions) amino acids which are involved in catalytic reactions) with in the active site. In addition, the substrate must with in the active site. In addition, the substrate must be precisely oriented to the catalytic groups. Once the be precisely oriented to the catalytic groups. Once the substrate is correctly positioned, a change in the substrate is correctly positioned, a change in the enzyme's conformation may result in a strained enzyme enzyme's conformation may result in a strained enzyme substrate complex. This strain helps to bring the substrate complex. This strain helps to bring the enzyme substrate complex into the transition stateenzyme substrate complex into the transition state. .

In general, the more tightly the active site can bind the In general, the more tightly the active site can bind the substrate while it is in the transition state, the greater substrate while it is in the transition state, the greater the rate of the reactionthe rate of the reaction

Proximity alone can only account for a five fold Proximity alone can only account for a five fold increase in activityincrease in activity

Without the proper orientation, little reaction can Without the proper orientation, little reaction can occur - the Sn2 attack is a good exampleoccur - the Sn2 attack is a good example

Lack of orientation is the key for an increased rate of Lack of orientation is the key for an increased rate of reactionreaction

Electrostatic EffectsElectrostatic Effects Recall that the strength of electrostatic interactions is related to the capacity of surrounding solvent molecules to reduce the attractive forces between chemical groups. Because water is largely excluded from the active site of most enzymes, the local dielectric constant is low. The charge distribution in the relatively anhydrous active site may influence the chemical reactivity of the substrate.

In addition, weak electrostatic interactions such as those between permanent and induced dipoles in both the reactive site and the substrate are believed to contribute to catalysis. A more efficient binding of substrate lowers the free energy of the transition state, which accelerates the reaction. Thus enzymes act by stabilizing the distribution of electrical charge in the transition states.

• Many inhibitors of enzymes mimic the transition state. The enzyme binds the inhibitor at a stable energy state - these inhibitors bind with a much higher affinity than the normal substrate.