Reaction mechanisms

Enzymes use many chemical techniques to encourage substrate reaction. These can be broadly grouped into three categories:

Acid-base catalysis.
Covalent catalysis.
Metal-ion catalysis.

Enzymes are very specific, reacting only with a very limited number of chemically similar substrates. This specificity is imposed by the geometry of the active site, which is normally situated in a cleft or pocket on the surface of the (globular) enzyme. The amino acids in the active site may be very widely spaced in the 1° structure of the protein but are brought close together by 2° and 3° folding: lysozyme digests bacterial cell walls, and is found in human tears, egg-white, etc. glutamate³⁵ and aspartate⁵² are involved in catalysis: note how far apart they are in the 1° structure.

Substrates are bound to the active site by multiple weak forces, such as hydrogen bonds, hydrophobic interactions, etc. Although enzyme specificity is high it is not perfect. There are two major ways of increasing substrate specificity. Enzymes make relatively weak bonds with their substrate: only those substrates able to make many of these bonds are sufficiently warped to enter the transition state. A few enzymes (DNA polymerase and aminoacyl tRNA synthetases) further increase specificity by proofreading after the reaction has occurred.

Type II restriction enzymes are endonucleases that cut at very specific sites in DNA. They are used by bacteria to destroy bacteriophage DNA and are useful tools in molecular biology. Restriction enzymes act as homodimers, hence they cut at palindromic sequences. EcoRV recognises the six base-pair palindrome:

5'–GAT|ATC–3'

3'–CTA|TAG–3'

and cuts where indicated by the | character, forming 'blunt ends'. These sites should occur with a frequency of about (¼)⁶, i.e. once in every 4096 bp, hence the enzyme must be about 5000 times better at cutting GATATC (it's cognate DNA) than any other random noncognate sequence.

Restriction enzymes bind indiscriminately to any DNA sequence but they only cleave one. The reason for this is that the enzyme forms hydrogen-bonds to the G and A upstream of the cleavage site, and this releases energy that is able to warp the DNA.

Reaction coordinate for cognate and noncognate DNA hydrolysis by EcoRV.
EcoRV cannot cut noncognate DNA because when noncognate DNA binds to the enzyme, it does not release sufficient energy to warp the DNA and bring it into the active sites of the enzyme.

Only the warped DNA is able to bind magnesium ions between itself and the enzyme.

Cognate DNA is much more warped by EcoRV than a noncognate ligand. Note warping of DNA helix (orange) between the two EcoRV monomers (pink and red) close to the magnesium ions (green spheres). Noncognate DNA is not warped and is not brought close to the active sites.

Magnesium also binds water molecules, and this brings them close to the phosphate backbone, where they can cause cleavage of the bond.

EcoRV works by binding water to magnesium and bringing this into close approximation to the DNA's phosphate backbone.
Mechanism of action: the magnesium ion is bound to the COOH groups of two aspartate residues, to the phosphate group of the DNA, and to three water molecules. Ones of these water molecules attacks the phosphodiester bond, cleaving the DNA backbone.

Aminoacyl tRNA synthetases join amino acids to tRNAs. These enzymes must be must be very accurate despite amino acids being rather similar in structure, otherwise translation of mRNA would lead to informational meltdown. They use a second binding step after the initial condensation of amino acid to tRNA. This acts as a proof-reading step.

Alanine is attached to tRNA by an aminoacyl synthetase.
tRNA^ala needs to be attached to alanine.

Accidental attachment of glycine to the alanine tRNA.
However, because alanine and glycine are so similar, glycine can be attached by accident.

Esterase activity of alanyl tRNA synthetase removes glycine.
Glycine is smaller than alanine, so the synthetase has a secondary esterase activity that removes erroneously attached amino acids from the tRNA.

The tRNA is large and easily distinguished, so there is only one error every 10 000 catalytic cycles. Amino acids are small and easily confused, and there is an error every 200 catalytic cycles, which is too many for translation to cope with and still produce workable proteins the majority of the time. So the enzyme has a second active site, which is smaller and has esterase activity. Only glycine fits in this esterase site, and is cleaved off.

The enzyme-substrate binding energy is used to lower the activation energy for the transformation of substrate to products. The active site is usually complementary to the transition state. For the enzyme proline racemase, pyrrole-2-carboxylic acid is a competitive inhibitor of proline racemase: this is because it is a planar analogue of the transition state.

Proline racemase is inhibited by a molecule that resembles the planar transition state.

Another interesting consequence of transition-state complementarity is that if we raise an antibody to a molecule resembling the transition state, this 'abzyme' often catalyses the reaction too, e.g. N-methyl-protoporphyrin IX is buckled, like haem is when Fe²⁺ is added by ferrochelatase. Antibodies to N-methyl-protoporphyrin IX have ferrochelatase-like activity.

Antibodies raised against a transition-state mimicking molecule can catalyse reactions.

Acid-base catalysis

Acid-base mechanisms rely on reactive groups on the side-chains of amino acids rearranging protons in the substrate. Acidic, alcoholic and basic amino acids are involved most often.

Amino acid	Group	pK_a	Functions
Aspartate	-COOH	3.9	Bind H⁺ and other cations
Glutamate	-COOH	4.5	Covalent bonding of acyl groups
Serine	-OH	13.0
Cysteine	-SH	8.5
Tyrosine	-OH	10.1	H-bond to substrates
Histidine	-NH₂⁺-	6.0	Bind H⁺
Lysine	-NH₃⁺	10.5	Bind anions
Arginine	-CH(NH₂)²⁺	12.5	Bind anions

An acid is a proton donor. A base is a proton acceptor.

RCOOH + H₂O ⇌RCOO⁻ + H₃O⁺

RNH₂ + H₂O ⇌RNH₃⁺ + OH⁻

Note that water can act as both an acid and a base. In each of the following examples, we will look at a reaction catalysed by a simple chemical species, then compare this to a similar mechanism involving an enzyme. The following shows the mechanism of base-catalysed hydrolysis of alanylalanine:

Alanylalanine hydrolysis is faster in the presence of bases.

Alanylalanine is degraded to two alanine molecules. Note the base (X⁻), which participates, but is unchanged by the end of the reaction. It aids hydrolysis by removing a proton from water to generate a stronger nucleophile (OH⁻). Note the charge-relay: electrons travel from the base to water to the C=O group.

Pancreatic RNAse also performs acid-base catalysis, but in this case, the protons are donated by histidine residues in the active site. Note the way the upper histidine removes a proton from the -OH of ribose, making it a sufficiently strong nucleophile to attack the phosphate group.

RNAse mechanism relies on a charge-relay between two histidine residues.

The pH profile of an enzyme gives some indication of amino acids in the active site, because amino acids such as histidine must be suitably ionised to bind substrates and/or abstract protons from them. As we decrease the pH, amine groups on lysine, histidine and arginine pick up protons. As we increase the pH, carboxylic acid groups on glutamic and aspartic acid will ionise to carboxylate groups.

pH affects the ionisation of side-chains in the enzyme.

Depending on the role of these groups in the catalysis, the acid or base form may be the one that is required. The pH optimum of an enzyme is a consequence of the changes in ionisation of active-site amino acids as the concentration of protons is increased or decreased. Here, glutamate (pK_a c. 4.5) must be present as the carboxylate anion, and lysine (pK_a c. 10) must be present as the ammonium cation. Remember that pK_a is the pH which gives 50% ionisation (or equivalently 50% of the pH-optimised rate of enzyme-catalysed reaction).

pH optima are the result of competing ionisations of enzyme side-chains.

The pH profile also gives an indication of the hydrophobicity of the cleft in which the active site resides: amino-acid side-groups find it difficult to ionise when surrounded by fat (it's difficult to get electrocuted in rubber boots). This increases the apparent pK_a for species that need to be present as anions (a higher pH - more hydroxide ions - is needed to pull the protons off the uncharged COOH group), and decreases it for those that need to be present as cations (a lower pH - more protons in solution - is needed to force a charge onto the uncharged NH₂ group.

Groups ionise less readily if they are in a hydrophobic cleft than in free solution.

Covalent catalysis

In covalent catalysis, a transient covalent bond is formed between the substrate and the enzyme. It is normally the result of nucleophilic attack by a group of the enzyme upon an electrophilic centre in the substrate, so this is often called nucleophilic catalysis too. The nucleophilicity of a substance is closely related to its basicity, however instead of abstracting a proton it forms a covalent bond.

Nucleophiles seek positive (nuclear) charge: -OH, OH⁻, -SH, -S⁻, -NH₂, =N:-
Electrophiles seek negative (electron) charge: H⁺, >C=O, >C=NH⁺-, Fe²⁺, Cu⁺

Alcoholic and basic amino acids are again involved, as they are the most polar.

Amino acid	Group
Threonine	-OH
Serine	-OH
Tyrosine	-OH
Cysteine	-SH
Histidine	-N=
Lysine	-NH₂
Arginine	-CH(NH₂)₂

Decarboxylation of acetoacetate is catalysed by primary amines. They form a covalent adduct together called a Schiff base (or imine), which is unstable and breaks down to form acetate ions, and regenerating the amine.

Acetoacetate decarboxylation is faster in the presence of amines, which form Schiff bases reversibly.

Acetylcholine esterase is an example of a serine hydrolase, a hydrolytic enzyme that contains serine in its active site:

AChE catalyses the hydrolysis of acetylcholine because it has a serine group that can act in the same way as water.

The reaction mechanism actually uses three amino acids - not just serine. Glutamine-327 hydrogen-bonds to histidine-440, orienting the ring so it interacts with serine-200. The histidine is able to act as a base, deprotonating the serine to generate a very nucleophilic alkoxide (–O-) group. It is really this group that attacks the ester bond: it is a much stronger nucleophile than an alcohol (–OH) group. This arrangement of serine, histidine and glutamate/aspartate is termed a catalytic triad and is a very common feature of hydrolases.

AChE contains a catalytic triad.

Chymotrypsin uses a similar catalytic triad mechanism: a histidine/aspartate pair in the active site (X) increases the nucleophilicity of a serine group by accepting a proton. This activated serine group then nucleophilically attacks the electrophilic carbonyl carbon in a peptide bond, forming a covalent bond and breaking the peptide bond.

Chymotrypsin mechanism involves nucleophilic attack.

Metal-ion catalysis

30% of all enzymes require a metal ion for catalytic activity. Some enzymes bind the (usually d-block) metal very tightly and are known as metalloenzymes. These metal ions are used to bind substrates, perform redox reactions, shield negative charges, etc. e.g. Fe, Cu, Zn, Mn, Co. Other enzymes bind them more loosely. These metal ions (usually s-block) are called activators and play a role in maintaining the structure of the protein rather than participating in the catalytic cycle. e.g. Na, K, Mg, Ca. The restriction enzyme example given above is a (slightly anomalous) example of metal-ion catalysis by an s-block metal (magnesium).

Metal ions undergo redox reactions, transferring electrons to and from substrates.
OIL-RIG: oxidation is the loss of electrons; reduction is the gain of electrons.

Zinc is bound to three histidines in carbonic anhydrase. Water is ionised to a hydroxide ion and this is stabilised by the Zn²⁺ ion. CO₂ then enters the active site and is attacked by OH⁻, forming a carbonate ion, which is then released, regenerating the enzyme.

Carbonic anhydrase binds carbon dioxide and water through a histidine-complexed zinc ion.

Test yourself

Why do aminoacyl tRNA synthetases require two active sites?
What are the catalytic roles of histidine in the enzymes we have discussed above?

Answers

Bibliography

Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 208-211. "Free energy is a useful thermodynamic function for understanding enzymes"
Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 243-254. "Proteases facilitate a fundamentally difficult reaction"
Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 254-259. "Carbonic anhydrases make a fast reaction faster"
Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 259-270. "Restriction enzymes perform highly specific DNA-cleavage reactions"
Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 276-282. "Aspartate transcarbamoylase is allosterically regulated by the end product of its pathway"
Lerner, R. A. and Tramontano, A. (1988). Catalytic antibodies. Scientific American 258:58-70

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