Chapter 7: Enzyme Reaction Mechanisms

7.1 How Enzymes Work

7.5 Ribozymes

7.1 How Enzymes Work

Reactions in solution that are not catalyzed are slow since charge development and separation occurs in the transition state. When bonds are made or broken, charged intermediates are often formed which are higher in energy than the reactants. Since the intermediate is higher in energy than the reactants, the transition state would be even higher in energy, and hence more closely resemble the charged intermediate. Anything that can stabilize the charges on the intermediate and hence the developing charges in the transition states will lower the energy of the transition state and catalyze the reaction. In this section will will investigate the mechanism underlying the catalysis by small molecules of chemical reactions. Presumably, biological macromolecular catalyst (like protein enzymes) will use similar mechanisms in their catalytic effects (which will be discussed in the next section).

Catalysts, including enzymes, can employ at least 5 different ways to stabilize transition states.

General Acid and Base Catalysis

Charge development in the TS can be decreased by either donation of a proton from general acids (like acetic acid or a protonated indole ring) to an atom such as a carbonyl O which develops a partial negative charge in the TS when it is attached by a nucleophile. Proton donation decreases the developing negative in the TS. Alternatively, a nucleophile such as water which develops a partial positive charge in the TS as it begins to form a bond to an electrophilic C in a carbonyl can be stabilized by the presence of a general base (such as acetate or the deprotonated indole ring). Proton abstraction decreases the developing positive charge





Metal Ion or Electrostatic Catalysis

A metal such as Cu2+ or Zn2+ can also stabilize the TS. The metal must be able to be bound to the charged intermediate and hence the TS. The tetrahedral oxyanion intermediate of the reaction of an electrophilic carbonyl C can interact with a metal if there is an O on an adjacent atom which can help coordinate the metal ion. This charge stabilization of the developing negative in the TS and the full negative in the intermediate is often called electrostatic catalysis. This method is likely to be found in many enzymes since nearly 1/3 of all enzymes require metal ions. A classic example of an enzyme using metal ion catalysis is carboxypeptidase A.


Metals can also act in a different way. They may coordinate a water and by further polarizing the H-O bond increase the acidity of the bound water. For instance, the water molecule in the pentammineaquacobalt(III) ion has a pKa of 6.6, compared to pure water, with a pKa of 15.7. (To calculate the latter, write the equilibrium expression for water: H2O + H2O <==> H3O+ + OH. Then write the Ka expression, as for a generic acid, which is [H3O+][ OH-]/ [H2O] = 10-14/55.5. The pKa is 15.7). The complexed hydroxide is a better nucleophile than bulk water. An example of an enzyme whose bound metal increases the nucleophilicity of water is carbonic anhydrase.



Covalent or Nucleophilic Catalysis

One way to change the activation energy of the reaction is to change the reaction mechanism in ways which introduces new steps with lower activation energy. A typical way is to add a nucleophilic catalyst which forms a covalent intermediate with the reactant. The original nucleophile can then interact with the intermediate in a nucleophilic substitution reaction. If the nucleophilic catalyst is a better nucleophile than the original nucleophile (usually water) then the reaction is catalyzed. The nucleophilic catalyst and the original nucleophile usually interact with a carbonyl C in a substitution reaction, initially forming the tetrahedral oxyanion intermediate.


If an amine is used as the nucleophilic catalyst, then the initial addition product (a carbinolamine) can become dehydrated, since the free pair of electrons on the N are more likely to be shared with the carbon to form a double bond than electrons from the original carbonyl O, which is more electronegative than the N). An imine or Schiff Base forms, with a pKa of about 7.


This is easily protonated to form a positively charged N at the former carbonyl O center. This serves as an excellent electron sink for decarboxylation reactions of beta-keto acids and illustrates an important point. Electrons in chemical reactions can be viewed as flowing from a source (such as a carboxyl group) to a sink (such as an nucleophilic carbonyl O or a positively charged N in a Schiff base).



In a subsequent section, we will discuss how protein enzymes use these same catalytic strategies. An intriguing question arises: how much of the structure of a large protein is really needed for catalysis? Much work has been directed to the development of small molecule catalysis mimetics of large protein enzymes. Just how small can you go in reducing the size of a protein and still get catalysis. One important feature of enzyme catalysis is that they catalyze reactions in which only one enantiomer is produced. That is, the synthesis is assymertric. This is typically a consequence of the asymmetric enzyme (itself chiral) binding only one enantiomer as a reactant and/or the imposition of steric restrictions on the possible reactions of the bound substrate. Recently, it has been show that L-Pro alone can act as such an assymetric catalyst in an aldol condensation reaction.



Intramolecular Catalysis (reference)

Consider the hydrolysis of phenylacetate. This reaction, a nucleophilic subsitution reaction, could be catalyzed by the addition to solution of the general base acetate, as described above. Since this reaction would double with the doubling of the solution acetate, the reaction is bimolecular (first order in reactant and catalyst). Now consider the same reaction only when the the general base part of the catalyst, the carboxyl group, is part of the reactant phenylacetate. Such a case occurs in the acetylated form of salicylic acid – i.e. aspirin. When the carboxy group is ortho compared to the acetylated phenolic OH, it is in perfect position to accept a proton from water, decreasing the charge development on the O in the transition state. The general base does not have to diffuse to the appropriate site when it is intramolecular with respect to the carbonyl C of the ester link. The rate of this intramolecular base catalysis is about 100 fold greater than of an intermolecular base catalyst like acetate. It is as if the effective concentration of the intramolecular carboxyl base catalyst is much higher due to its proximity to the reaction site.

Another type of reactions involving a carboxyl group (in addition to simple proton transfer) is when the negatively charged carboxyl O acts as a nucleophile and attacks an electrophilic carbonyl carbon. When the carbonyl is part of an ester, the carboxyl group engages in a nucleophilic substitution reaction, expelling the alcohol part of the ester as a leaving group. The remaining examples below consider the nucleophilic (carboxyl) substitution on phenylesters, with phenolate as the leaving group. The reactions in effect transfer an acyl group to the carboxyl group to create an anhydride.

First consider acyl transfer with aspirin derivatives. Aspirin, as you know, contains a carboxyl group ortho to an ester substitutent. Hence the carboxyl group can act as a nucleophile and attack the carbonyl carbon of the ester in a nucleophilic substitution reaction. The net effect is to transfer the acetyl group from the phenolic OH to the carboxyl group converting it to an anhydride. This is an intramolecular reaction. Compare this reaction to a a comparable bimolecular reaction shown below.


The first order rate constant of the intramolecular transfer of the acetyl group to the carboxyl group, k1 = 0.02 s-1. The analogous bimolecular reaction rate constant k2~ 10-10 M-1s-1. Dividing k1/k2 gives the relative rate enhancement of the intramolecular over the intermolecular reaction. With units of molarity, this ratio can be interpreted as the relative effective concentration of the intramolecular nucleophile. This makes the effective concentration of the carboxylate in the aspirin derivative 2 x 107 M.

Now consider the cleavage of phenylacetate using acetate as the nucleophile. The products are acetic anhydride and phenolate. This is a bimolecular reaction (a slow one at that), with a bimolecular rate constant, k2 which I will arbitrarily set to 1 for comparison to some similar reactions.


Now consider a monoester derivatives of succinic acid – phenyl succinate – in which the free carboxyl group of the ester attacks the carbonyl carbon of the ester derivative.


If you assign a second order rate constant k2 = 1 M-1s-1 to the analogous intermolecular reaction of acetate with phenylacetate (as described above), the first order rate constant for the intramolecular reaction of phenylsuccinate is 105 s-1. The ratio of rate constants, k1/k2 = 105 M. That is it would take 105 M concentration of acetate reacting with 1 M phenylacetate in the first bimolecular reaction to get a reaction as fast as the intramolecular reaction of phenylsuccinate. An even more sterically restricated bicyclic phenylcarboxylate shows a k1/k2 = 108 M.


Another example is anhydride formation between two carboxyl groups. The ΔGo for such a reaction is positive, suggesting an unfavorable reaction. Consider two acetic acid molecules condensing to form acetic anhydride. For this intermolecular reaction, Keq = 3×10-12 M-1. Now consider the analogous intramolecular reaction of the dicarboxylic acid succinic acid. It condenses in an intramolecular reaction to form succinic anhydride with a Keq = 8×10-7 (no units). The ratio Keq-intra/Keq inter = 3 x 105 M. It is as if the effective concentration of the reacting groups. because they do not have to diffuse together to react, is 3 x 105 M.

How does this apply to enzyme catalyzed reaction? Enzymes bind substrates in physical steps which are typically fast. The slow step is chemical conversion of the bound substrate, which is effectively intramolecular. These three kinds of reactions, intermolecular, intramolecular, and enzyme-catalysed can be broken down into two hypothetical steps, a binding followed by catalysis.

Figure: three kinds of reactions, intermolecular, intramolecular, and enzyme-catalysed

If the rate constants for the chemical steps are all identical, the advantage of the intramolecular and enzyme-catalyzed reaction over the intermolecular reaction is KINTRA/KINTER and KENZ/KINTER, respectively.

The advantage of intramolecular reactions can be seen by studying the Ca-EDTA complex. Calcium in solution exists as a octahedrally coordinated complex with water occupying all the coordination sites. EDTA, a multidentate ligand, first interacts through one of its potential six electron donors to Ca in a reaction which is entropically disfavored from the the Ca-EDTA perspective, although one water is released. Once this first intramolecular complex is formed, the rest of the ligands on the EDTA rapidly coordinate with the Ca and release bound water. The former is no longer entropically disfavored since it is now an intramolecular process while the later is favored through the release of the remaining five water molecules.


We modeled the catalytic advantage offered by intramolecular reaction in terms of a dramatic increase in the effective concentration of reactants, which sometimes reached levels of 108 M. Another way is to look at entropy changes associated with dimer formation. The table below shows that an intramolecular reaction is favored over an intermolecular reaction since in the latter, significant decreases in translational and rotation entropy result.

Translational, Rotational, and Internal Entropies
for Dimer Formation: A + B <=> A-B (cal/K.mol)

System A B A-B ΔS
S trans 30 30 30 -30
S rot 20 20 20 -20
S int 5 5 20 +10
Gas -> Solution -10 -10 -15
S sol 45 46 55 -35 (Correspond to 108-109 M)



Transition State Stabilization

Edit section

Linus Pauling postulated long ago that the only thing that a catalyst must do is bind the transition state more tightly than the substrate. That this must be the case can be seen from the diagram below, which shows how S and S* (the transition state) can react with E to form a complex which then proceeds to product, or can go to product in the absence of E. From this diagram, it should be evident that c – a = d -b, where a is the ΔGo for the binding of S to E, and b is the ΔGo for the binding of S* to E. For an enzyme to be a catalyst the activation energy for the reaction in the presence of E, d, must be less than in the absence of enzyme, c. Therefore c-d = a-b > 0. Since ΔGo = -RTln Keq, Keq for binding of S* to E is greater than for S binding to E.


The stability of the transition state also affects the reaction kinetics (which makes sense given that the activation energy clearly affects the speed of a reaction). As you probably remember from organic chemistry, SN2 reactions are slow when the central atom where the substitution will occur is surrounded by bulky substitutents. (Sterics once again.) We discussed this in context to nucleophiliic substitution on a sp2 hybridized carbonyl carbon in carboxylic acid derivatives versus on a sp3 hybridized phosphorous in phosphoesters and diesters. The explanation for this phenomena has usually been attributed to hindered access of the central atom caused by bulky substituents (intrinsic effects). Is this true? Studies on SN2 reactions of methylchloroacetonitrile and t-butylchloroacetonitrile (with the reagent labeled with 35Cl) using 37Cl- as the incoming nucleophile in the gas phase shown that the more hindered t-butyl derivative’s activation energy was only 1.6 kcal/mol higher than the methyl derivative, but in aqueous solution, the difference is much greater for comparable reactions. They attributed the differences to solvation effects of the transition state. The bulkier the substituents on the central atom, the more difficult it is to solvate the transition state since water can’t reorient around it as well. In effect there is steric hindrance for both reactant and solvent.

Abyzmes – Antibody Catalysis

What does it take for a macromolecule (M) to be a catalyst – an enzyme. It seems the minimum criterion are:

  • M binds a reactant
  • M binds the transition state more tightly than the substrate

Anything above these is just “icing on the cake”. If different functional group are present in the “active” site of the enzyme that would allow electrostatic, intramolecular, covalent, general acid and/or base catalysis, the better the catalyst.

Linus Pauling recognized the two key factors decades ago. He made the following hypothesis: Antibody molecules (immune system proteins that bind foreign molecules) that can be made to bind to transition state analogs of a substrate, should also presumably catalyze the conversion of substrate, through the transition state, to product. About a decade ago, his prediction was verified. Lerner et al. made a transition state analog of an ester. When an ester is hydrolyzed, the sp2 hybridized carbonyl carbon is converted to an sp3 hybridized center in the intermediate, with the carbonyl oxygen becoming an oxyanion. The transition state presumably looks more like this unstable intermediate (sp3, oxyanion). Lerner synthesized a phosphonate, an ester mimic with a sp3 hybridized phosphorous replacing the carbonyl C. It also has a negatively charged oxygen as does the intermediate for the ester. This phosphonate ester is very resistant to hydrolysis. When injected into a mouse (after first being covalently attached to a carrier protein so the small molecule becomes “immunogencic”), the mouse makes a protein antibody which binds to the phosphonate. When the corresponding carboxylic acid ester is added to the antibody, it is cleaved with nominal kcat and Km values. Site specific mutagenesis can then be done to make it an even better catalyst! The antibody enzymes have been called abzymes. The structure below shows how phosphonamides act as transition state analogs as well.


Jmol: Updated Immunoglobulin 48G7 Germline Fab Antibody Complexed With Hapten 5-(Para-Nitrophenyl Phosphonate)-Pentanoic AcidJmol14 (Java) | JSMol (HTML5)

Transition state theory can be used to more clearly quantify the relationships described in the graphical analysis above. This analysis will use the equilibrium constant (in contrast to the last two chapters which used dissociation constants to characterize macromolecule, receptor, and enzyme binding to ligand). Let assume that a substrate S is in equilibrium with its transition state S‡. Hence Keq = [S‡]/[S]. The following reaction can be written: S –> S‡ –> P. Based on our previous kinetic analysis and experience in writing differential equations, dP/dt = k1[S‡]. By analogy, enzyme bound S (ES) can be converted to (ES‡) and then on to product as shown in the following chemical equation:
E + S <—-> ES –> ES‡ –> E + P.

For the non-enzyme catalyzed reaction, transition state theory can be used to show that the first order rate constant k1= kT/h where k is the Boltzman’s constant, T is the Kelvin temperature, and h is Planck’s constant. Hence, using Keq = [S‡]/[S], equation 1 can be derived

1. TS Theory Eq 1

where kn (hereafter written as kN) =(kT/h)K‡ is the effective first order rate for the non-catalyzed rate. Now lets create a more complicated linked equilibrium showing the same reaction in the presence of an enzyme.


Remember that the K values for this analysis are equilibrium constants not dissociation constants. Note two important equilibrium constants, KS, the equilibrium constant for the binding of free S to E, and KT, the equilibrium constant for the binding of free S‡ to E (assuming that free S‡ could bind to E before it converted to product). As we have seen for linked equilibrium before, since the Keq values are related to the standard free energy changes which are state functions, the sum of the standard free energies going from E + S to ES‡ (by either the top or bottom paths) are path independent so the products of the Keq for the top path are equal to those for the bottom paths. This gives the following equation:


The right hand side is the ratio of the effective first order rate constant for conversion or ES‡ –> E + P, kE dvided by the rate constant for the conversion of S‡ –> P for the noncatalyzed rate, kN. The final ratio of rate constants can be derived from the simple relationship that kx=(kT/h)K‡x where x is either N (non catalyzed) or E (enzyme catalyzed). Equation 2 states that the equilibrium constant for the binding of S‡ to E, KT, is greater than the equilibrium constant for the binding of S to E, KS (as kE > kN). KT/KR ranges from 108 – 1014. Given common values for the equilibrium constant for binding of S to E (103 – 105 M-1) which is equivalent to dissociation constant values Kd = 10 uM -1 mM, the calculated value of KT = 1015 M-1 which gives a dissociation constant for the enzyme and transition state of Kd = 10-15 M (1 femtomolar). This is as tight as one of the highest affinity binding interactions in the biological world, the binding of avidin and biotin. As we noted in Chapter 5A, assuming that the second order rate constant for avidin/biotin binding and as shown above for E/S‡ is diffusion controlled (about 108 M-1s-1), the off rate for the avidin-biotin or ES‡complex is 10-7 s-1, equivalent to a half life of the complex of 80 days. It doesn’t get much tighter than that.

The figure below represent an image of an enzyme and three different molecules, 1-3, that could bind to it. Using the analysis above, which molecule do you think represents substrate? Transition state? Product?

Figure: 3D model for binding substrate, transition state, and product to an enzyme

Animated Version

TS binding 3D


7.5 Ribozymes

Any molecule that displays any of the catalytic motifs seen in the earlier chapters (general acid/base catalysis, electrostatic catalysis, nucleophilic catalysis, intramolecular catalysis, transition state stabilization) can be a catalyst. So far we have examined only protein catalysts. These can fold to form unique 3D structures which can have active sites with appropriate functional groups or nonprotein “cofactors” (metal ions, vitamin derivatives) that participate in catalysis. There is nothing special about the ability of proteins to do this. It is now known that RNA, which can form complicated secondary and tertiary structures as seen in the 3D image of the ribozyme from Tetrahymena thermophila, can as well.

Figure: ribozyme from Tetrahymena thermophila,

RNA molecules that act as enzymes are called ribozymes. This property of some RNA’s was discovered by Sidney Altman and Thomas Czech, who were awarded the Nobel Prize in Chemistry in 1989. In contrast to protein enzymes which are true catalysts in that they are used over again, this is an example of a single use ribozyme. Other ribozymes are true catalysts and can carry out RNA slicing by transesterification (splicesome) and peptidyl transfer (in ribosomes). The mechanisms of catalysis of the hepatitis delta virus ribozyme include general acid/base catalysis.

Figure: mechanisms of catalysis

ribozyme mechanism

The hairpin ribozyme from satellite RNAs of plant viruses is 50 nucleotides long, and can cleave itself internally, or , in a truncated form, can cleave other RNA strands in a transesterification reaction. The structure consists of two domains, stem A required for binding (self or other RNA molecules) and stem B, required for catalysis. Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases (which are splayed apart) when the 2′ OH on the A attacks the phosphorous in the phosphodiester bond connecting A and G to form an pentavalent intermediate.

Figure: Self-cleavage in the hairpin ribozyme

Rupert & Ferr�-D’Amar� (2001) solved the crystal structure of a hairpin ribozyme with a non-cleavable substrate analog containing a in which a 2′-OCH3 was substituted for the nucleophilic 2′-OH group. See the applet below.

A recent study by Rupert et al. (2002) shows that A38 in Stem B appears to be able to interact with the products (the cleaved A now in the form of a cyclic phosphodiester with itself) and the departing G, and also with a transition state pentavalent analog of the sessile A-G bond in which the phosphodiester linking A and G in the substrate is replaced with a pentavalent vanadate bridge between A and G. However, A 38 does not appear to react with the sessile A -G groups in the normal substrate, indicating that the main mechanism used by this ribozyme is transition state binding. Since RNA molecules have fewer groups available for acid/base and electrostatic catalysis (compared to protein enzymes), ribozymes, presumably the earliest type of biological catalyst, probably make more use of transition state binding as their predominant mode of catalytic activity.

Figure: Active Site of Hairpin Ribozyme: Transition State Binding

Recently, the crystal structure of a purple bacterium group I self-splicing intron (which catalyze the removal of itself) interacting with both exons in a state prior to their ligation was determined (Adams, P. et al.). The structure shows both exons in close proximity. Nucleophilic attack of the 3’OH of the 5′ exon on a distorted phosphate at the intron-3′-exon junction. Two metal ions reside on either side of the labile phosphodiester bond at the intron-3’exon junction, and are held in place by 6 phosphates.

A novel use of ribozymes was recently reported by Winkler et. al. They discovered that the 3′ end of the mRNA of the gene glmS (from Gram-positive bacteria) which encodes an amidotransferase (catalyzing the formation of glucosamine-6-phosphate from glutamine and fructose-6-phosphate) is a ribozyme. A glucosamine-6-phosphate binding site in the ribozyme (3′ end of the mRNA) binds this sugar, inducing autocleavage of the ribozyme. This inhibits, by an uncertain mechanism, the formation of the amidotransferase from the remaining part of the mRNA. This mechanism of regulation of gene expression through ribozyme activity might prove to be common.

In order for RNA molecules to have acted as both catalysts and carriers of genetic information in the original RNA world, RNA must have the potential to self replicate. Shechner et al. looked at the core structure of a class I ligase ribozyme able to polymerize RNA. This ligase was synthesized and enhanced through The tripod structure allows more RNA solvent interaction then often found in ribozymes. Several common structural motifs were also found to be present including the GNRA-triloop, uridine-turn and the frameshifting pseudoknot motifs. GNRA-triloop and uridine-turn motifs are short thermodynamically stable segments that cap the end of the helical regions. Varieties of these are found in many RNA structures although they don’t seem to be necessary for activity. The frameshifting pseudoknot motif is almost identical in structure to small viral ribosomal frameshifting pseudoknots and is adjacent to a new motif named “A-minor triad”. The A-minor triad is responsible for the coordination with Mg2+, although only when inserted in-between certain sequences. Triphosphoguanosine (called G1) at the 5’ terminus of the ribozyme acts as the electrophile for the RNA ligation reaction. Other segments of the ribozyme, such as the J1/2, have highly specific contacts which orient G1 and allow the RNA to fold more efficiently. Two residues, cytosine 12 (C12) and uracil 48 (U48) bind to Mg2+. Other sections of the RNA also promote specificity to assist in the replication process. A model for catalysis and the transition state of the ribozyme polymerase is similar to that for protein RNA polymerases, as shown in the figure below

Figure: Comparison of Transition State Models of Ribozyme and Protein RNA Polymerases (after Schechner et al)

A divalent Mg2+ in the active site of the ribozyme enhances the nuclophilicity of the 3-OH on the primer, which attached the the terminal phosphate of the G(1)TP substrate to form a pentavalent intermediate. The Mg cation is stabilized by oxygens on P 29 and 30 of the ribozyme. The Mg ion also stabilizes the developing charge in the transition state and in the charge in the intermediate. Stabilization of analogous divalent cations in the protein polymerase occurs through Asp side changes in the protein.

Jmol: Updated Self-splicing Group I intron with both exons Jmol14 (Java) | JSMol (HTML5)

Jmol: Updated L1 Ligase Ribozyme Jmol14 (Java) | JSMol (HTML5) Not done; Fix

The Biggest Ribozyme – The Ribosome

Protein synthesis from a mRNA template occurs on a ribosome, a nanomachine composed of proteins and ribosomal RNAs (rRNA). The ribosome is composed of two very large structural units. The smaller unit (termed 30S and 40S in bacteria and eukaryotes, respectively) coordinates the correct base pairing of the triplet codon on the mRNA with another small adapter RNA, transfer or tRNA, that brings a covalently connected amino acid to the site. Peptide bond formation occurs when another tRNA-amino acid molecule binds to an adjacent codon on mRNA. The tRNA has a cloverleaf tertiary structure with some intrastranded H-bonded secondary structure. The last three nucleotides at the 3′ end of the tRNA are CpCpA. The amino acid is esterified to the terminal 3’OH of the terminal A by a protein enzyme, aminoacyl-tRNA synthetase.

Covalent amide bond formation between the second amino acid to the first, forming a dipeptide, occurs at the peptidyl transferase center, located on the larger ribosomal subunit (50S and 60S in bacteria and eukaryotes, respectively). The ribosome ratchets down the mRNA so the dipeptide-tRNA is now at the the P or Peptide site, awaiting a new tRNA-amino acid at the A or Amino site. The figure below shows a schematic of the ribosome with bound mRNA on the 30S subunit and tRNAs covalently attached to amino acid (or the growing peptide) at the A and P site, respectively.

Figure: Prokaryotic Ribosme – P and A sites

A likely mechanism (derived from crystal structures with bound substrates and transition state analogs) for the formation of the amide bond between a growing peptide on the P-site tRNA and the amino acid on the A-site tRNA is shown below. Catalysis does not involve any of the ribosomal proteins (not shown) since none is close enough to the peptidyl transferase center to provide amino acids that could participate in general acid/base catalysis, for example. Hence the rRNA must acts as the enzyme (i.e. it is a ribozyme). Initially it was thought that a proximal adenosine with a perturbed pKa could, at physiological pH, be protonated/deprotonated and hence act as a general acid/base in the reaction. However, none was found. The most likely mechanism to stabilize the oxyanion transition state at the electrophilic carbon attack site is precisely located water, which is positioned at the oxyanion hole by H-bonds to uracil 2584 on the rRNA. The cleavage mechanism involves the concerted proton shuffle shown below. In this mechanism, the substrate (Peptide-tRNA) assists its own cleavage in that the 2’OH is in position to initiate the protein shuttle mechanism. (A similar mechanism might occur to facilitate hydrolysis of the fully elongated protein from the P-site tRNA.) Of course all of this requires perfect positioning of the substrates and isn’t that what enzymes do best? The main mechanisms for catalysis of peptide bond formation by the ribosome (as a ribozyme) are intramolecular catalysis and transition state stabilization by the appropriately positioned water molecule.

Figure: Mechanism Peptide Bond Formation by the Ribosome

The crystal structure of the eukaryotic ribosome has recently been published (Ben-Shem et al). It is significantly larger (40%) with mass of around 3×106 Daltons. The 40S subunit has one rRNA chain (18) and 33 associated proteins, while the larger 60S subunit has 3 rRNA chains (25S, 5.8S and 5S) and 46 associated proteins. The larger size of the eukaryotic ribosome facilitates more interactions with cellular proteins and greater regulation of cellular events. The Jmol structure of a bacterial 70S ribosome showing mRNA and tRNA interactions is shown below.