Enzyme catalysis

Enzyme catalysis is the catalysis of chemical reactions by specialized proteins, enzymes. Catalysis of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions.

The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative reaction route and by stabilizing intermediates the enzyme reduces the energy required to reach the highest energy transition state of the reaction. The reduction of activation energy (ΔG) increases the number of reactant molecules with enough energy to reach the activation energy and form the product.  

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Induced fit

  The favored model for the enzyme-substrate interaction is the induced fit model.^[1] This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.

Catalysis by induced fit

  The advantages of the induced fit mechanism arise due to the stabilising effect of strong enzyme binding. There are two different mechanisms of substrate binding; uniform binding which has strong substrate binding, and differential binding which has strong transition state binding. The stabilizing effect of uniform binding increases both substrate and transition state binding affinity and differential binding increases only transition state binding affinity. Both are used by enzymes and have been evolutionarily chosen to minimize the ΔG of the reaction. Enzymes which are saturated, ie. have a high affinity substrate binding, require differential binding to reduce the ΔG, whereas largely substrate unbound enzymes may use either differential or uniform binding.

These effects have led to most proteins using the differential binding mechanism to reduce the ΔG, so most proteins have high affinity of the enzyme to the transition state. Differential binding is carried out by the induced fit mechanism - the substrate first binds weakly, then the enzyme changes conformation increasing the affinity to the transition state and stabilizing it, so reducing the activation energy to reach it.

It is important to clarify, however, that the induced fit concept cannot be used to rationalize catalysis. That is, the chemical catalysis is defined as the reduction of ΔG^‡ (when the system is already in the ES^‡) relative to ΔG^‡ in the uncatalyzed reaction in water (without the enzyme). The induced fit only suggests that the barrier is lower in the closed form of the enzyme but does not tell us what the reason for the barrier reduction is.

Mechanisms of transition state stabilization

These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the reaction's transition state, by providing an alternative chemical pathway for the reaction. There are six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism:

Catalysis by bond strain

This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater than to the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the energy difference between the substrate and transition state and helping catalyze the reaction.

However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization effect ^{[2] [3]}. Furthermore, enzymes are very flexible and they cannot apply large strain effect ^[4].

In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate residues in the active site.

Catalysis by proximity and orientation

This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together. This reduces the entropy of the reactants and thus makes reactions such as ligations or addition reactions more favorable, there is a reduction in the overall loss of entropy when two reactants become a single product.

This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzyme gives the reaction intramolecular character, which gives a massive rate increase.

However, the situation might be more complex, since modern computational studies have established that traditional examples of proximity effects cannot be related directly to enzyme entropic effects ^{[5] [6] [7]}. Also, the original entropic proposal ^[8] has been found to largely overestimate the contribution of orientation entropy to catalysis ^[9].

Catalysis Involving Proton donors or acceptors

See also: Protein pKa calculations

Proton donors and acceptors, i.e. acids and bases, may donate and accept protons in order to stabilize developing charges in the transition state. This typically has the effect of activating nucleophile and electrophile groups, or stabilizing leaving groups. Histidine is often the residue involved in these acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept and donate protons.

Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa. This alteration of pKa is possible through the local environment of the residue.

The pKa is can be modified significantly by the environment, to the extent that residues which are basic in solution may act as proton donors, and vice versa.

It is important to clarify that the modification of the pKa’s is a pure part of the electrostatic mechanism ^[3]. Furthermore, the catalytic effect of the above example is mainly associated with the reduction of the pKa of the oxy anion and the increase in the pKa of the histidine, while the proton transfer from the serine to the histidine is not catalyzed significantly since, it is not the rate determining barrier ^[10].

Electrostatic catalysis

Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come from acidic or basic side chains found on amino acids such as lysine, arginine, aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.

Systematic computer simulation studies established that electrostatic effects give, by far, the largest contribution to catalysis ^[3]. In particular, it has been found that enzyme provides an environment which is more polar than water, and that the ionic transition states are stabilized by fixed dipoles. This is very different from transition state stabilization in water, where the water molecules must pay with "reorganization energy" ^[11] in order to stabilize ionic and charged states. Thus, the catalysis is associated with the fact that the enzyme polar groups are preorganized ^[12].

Covalent Catalysis

Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed. Schiff base formation using the free amine from a lysine residue is another mechanism, as seen in the enzyme aldolase during glycolysis.

It is important to clarify that covalent catalysis does correspond in most cases to simply the use of a specific mechanism rather than to true catalysis ^[3]. For example, the energetics of the covalent bond to the serine molecule in chymotrypsin should be compared to the well-understood covalent bond to the nucleophile in the uncatalyzed solution reaction. A true proposal of a covalent catalysis (where the barrier is lower than the corresponding barrier in solution) would require, for example, a partial covalent bond to the transition state by an enzyme group (e.g., a very strong hydrogen bond), and such effects do not contribute significantly to catalysis.

Quantum Tunneling

These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of "through the barrier" mechanisms (quantum tunneling). Some enzymes operate with kinetics which are faster than what would be predicted by the classical ΔG^‡. In "through the barrier" models, a proton or an electron can tunnel through activation barriers. ^[13][14] Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine dehydrogenase.^[15]

Interestingly, quantum tunneling does not appear to provide a major catalytic advantage, since the tunneling contributions are similar in the catalyzed and the uncatalyzed reactions in solution ^{[14] [16] [17]}.

Examples of catalytic mechanisms

In reality, most enzyme mechanisms involve a combination of several different types of catalysis.

Triose phosphate isomerase

Triose phosphate isomerase (EC 5.3.1.1) catalyses the reversible interconvertion of the two triose phosphates isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.

Trypsin

Trypsin (EC 3.4.21.4) is a serine protease that cleaves protein substrates at lysine and arginine amino acid residues.

Aldolase

Aldolase (EC 4.1.2.13) catalyses the breakdown of fructose 1,6-bisphosphate (F-1,6-BP) into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP).

References

Further reading

• Alan Fersht, Structure and Mechanism in Protein Science : A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman, 1998. ISBN 0-7167-3268-8