ENZYMES - INTRO

ENZYMES – AN INTRODUCTION

What is an enzyme – in the broadest definition of the word?

An enzyme is a protein that catalyzes reactions that would proceed very slowly under the bodily conditions. Enzymes are incredible in their ability to speed up biological processes, in their specificity for their substrates and the fact that they function even in aqueous environments – few synthesized catalyzers share these properties.

Enzymes are essential to all living organisms, hence it is no surprise that many inheritable deceases are caused by enzyme deficiency.

Explain the terms cofactor, coenzyme and prostetic group!

Cofactor = typically ions, for instance Mg²⁺. Some enzymes don't need a cofactor at all, whereas others are completely dependent on them.

Coenzyme = a coenzyme is by definition a more complex organic or metalloorganic molecule (i.e. NADH) that are transient carriers of specific atoms or functional groups!

Coenzymes are often derived from vitamins.

Note that some enzymes need both cofactor/s and coenzyme/s to function!

Prosthetic group = a cofactor or a coenzyme that is very tightly bound to the enzyme, sometimes even covalently so.

What is an enzyme combine with its cofactor/coenzyme/prosthetic group called? What is the enzyme called without these features?

With = holoenzyme (thus – a functional enzyme)

Without = apoenzyme.

Enzymes are always proteins – whit one exception, which is?

RNAses. They consists of RNA molecules.

How are enzymes classified?

Through the reactions they catalyze.

Oxidreductases = transfer electrons (hydride ions or H atoms)

Tranferases = Group transfer reactions.

Hydrolases = Hydrolysis reactions (transfer of functional groups to water)

Lyases = Addition of groups to double bonds OR formation of double bonds by removal of groups.

Isomerases = Transfer of groups within molecules to yield isomeric forms.

Ligases = Formation of S-C, C-C, C-O or C-N bonds by condensation coupled to cleavage of ATP or a similar co-factor.

Why would biological reactions be slow without enzymes?

Because most biological molecules are actually stable in neutral pH and temperature that occurs in the cell. Furthermore many reactions in the cell, such as the collision of molecules with resulting reaction, would be highly unlikely to occur in an environment without enzymes.

What is the distinguishing feature of an enzymatic reaction?

That the enzyme provides an alternative environment in which a reaction can proceed at higher velocity!It is VERY, VERY important to understand that an enzyme do NOT affect the equilibrium, it only causes the reaction to reach the equilibrium faster. This is easy enough to understand if one consideres Le Chateliers principal once again : speeding up the process at one side of the arrow also speeds the process on the other side of the arrow.

What is the starting point for an enzymatic reaction?

The GROUND STATE.

What is “the transition state “?

The point at which substrate to product is just as likely as product to substrate. “The top of the energy hill”.

Define activation energy!

It is the difference between the ground state and the transition state.

What does this term mean? DG'°

DG' = the standard free energy change at pH =7!

what is the effect of a high activation energy?

Of course a slow reaction rate. See illustration below. The higher the hill... etc.

How can reaction rates be increased without enzymes?

Through high pressure or high temperature.

What is a rate-limiting step?

The step in a series of enzymatic reactions that has the highest activation energy.

How are equilibria and DG'° linked to each other and how is this expressed mathematically?

An equilibrium in its simplest form is expressed : S ↔ P. The equilibrium constant, Keq, is expressed : K´eq = [P]/[S]. In thermodynamics the relationship between Keq and DG'° is expressed :

DG'° = -RT ln K´eq

R = the gas constant; 8,315 J/mol

T = 298 K

The important thing to remember is : a large, negative DG'° reflects a favorable reaction – however it does NOT reflect the rate of the reaction! A large, negative DG'° can very well reflect a reaction that proceeds extremely slow.

What is the rate equation for the reaction S → P? What kind of reaction is it?

It is a first order reaction, since it is only dependent on [S].

It is expressed as V (the rate) = k [S]

What does the factor “k” tell us?

For instance, if the factor k is 0,05 /s, then 5% of the S molecules will turn into P molecules in 1 second. In a case of k = 2000/s , the reaction will of course proceed much faster.

What is the rate equation for the reaction : S1 + S2 = P?

This is a second order rate reaction, which translates to : V = k [S1] [S2]

How are the rate constant and activation energy mathematically related?

Through the equation : k = kT/h ∙ e^{- DG*/RT}

k denotes the Boltzmann constant in this case, h Planck´s constant and DG* is the activation energy.

So; what does this equation actually tell us? If you study it a bit, you'll see that it means that the rate constant k and DG* are inverse and exponential! In other words : a low activation energy leads to a faster reaction! And vice versa.

This is what enzymes do; they blower the activation energy! But how...?

What is the source of energy that enzymes use to lower the activation energy? And how can enzymes be so specific?

Binding energy ( DGB ) is the main source of energy used by enzymes to lower the activation energies for reactions. Binding energy is provided by covalent interaction between the substrate and the enzyme, and usually occur in the active site.

Energy is also derived from non-covalent bonds formed between the enzyme and the substrate.

Much energy is thus released through the energy released in the bond formations between substrate and enzyme. These bonds also provide specificity.

Weak interactions are optimized in the transition state – what does this mean?

It means that the enzyme and substrate are not complementary in them selves (which was previously believed and called “the lock and key”-model). An enzyme completely complementary to its substrate would be a poor enzyme! Because it would only stabilize the substrate, not “force” it to bend or change to fit the active site, thus resulting in a product.

The picture above is an excellent illustration of how the substrate must reach transition state in order to fit into the active site, and hence change into products in the process. Had the substrate been complementary to the active site to begin with, it would not have been forced to change!

How is specificity achieved?

Specificity is a definition om an enzyme´s ability to discriminate between its substrate and other molecules. Specificity is achieved by many different interactions; for instance hydrogen bonding between a Glu residue in the enzyme and a hydroxyl group in the substrate will not occur if the substrate lacks the functional group in question.

What different obstacles must be conquered in order for a reaction to occur?

Entropy ( freedom of motion) must be overcome – molecules that don't collide will not react with each other.

Water molecules surrounding the substrate due to the aqueous environment must be removed.

Molecules must be properly arranged in order for a reaction to occur.

The catalytical groups in the active site must also be properly aligned.

How are the obstacles listed above overcome?

All of them are overcome by binding energy. Entropy reduction is simply a result of substrates being bound to the enzyme.

Desolvation (removal of water) is another benefit provided by weak bond formations.

What does “induced fit” mean?

It is the change the enzyme itself undergoes as a result of substrate binding. This happens in most cases, but there are, of course, exceptions. Induced fit may affect the active site – or the entire enzyme. The “aim” is to bring the catalytical residues in place for a catalytical reaction – thus the conformational change makes the enzyme more effective!

Describe acid-base catalysis!

Acid-Base catalysis is a way to circumvent that charged intermediates goes back to their original forms as reactants. This is of course crucial, as no reaction will reach “closure” if the intermediates constantly go back to their original forms. In order to stabilize the intermediates, protons can be removed from or given to the intermediates. Acid-base catalysis is extremely common and occurs in the vast majority of enzyme catalyzed reactions. See example below.

What does specific acid-base catalysis mean?

It means that only H3O+ or OH- from water molecules are used in the process described above!

What does general acid-base catalysis mean?

That water is not enough – and hence that other molecules have to provide H3O+ or OH- , namely amino acids in the active site, especially : Glu, Asp, Lys, Arg, Cys, His, Ser, Tyr.

Describe covalent catalysis!

It means that a transient covalent bond is formed between the4 enzyme and the substrate.

Consider for instance the following reaction :

A-B →^H2O A + B , cleaved by a water molecule (hydrolysis).

In this case a nucleophilic group is required, X:

A-B + X: → A-X + B →^H2O → A + B + X:

The nucleophilic group provides an alternative path for the reaction. This path will only be used, however, if it has a lower activation energy than the uncatalysed pathway.

Describe metal ion catalysis!

Metal ions are sometimes completely necessary for the reaction to proceed. They may help to orient the substrate correctly or stabilize charged groups. They can also provide weak bond energy or participate in redox-catalysis.

MOST ENZYMES USE SEVERAL CATALYTICAL “METHODS” COMBINED!

Please do comment or feel free to ask me something if anything is unclear.