The chemical mechanisms of catalytic triad enzymes have been elucidated mainly from studies carried out on members of the serine protease family, but much of the mechanism can be extrapolated to the αβ hydrolases because of their very similar
catalytic triad layout. The following section describing the enzyme mechanism is therefore mainly based around what is known from serine hydrolase research. The chemistry of the mechanism, described in the following sections, is shown in Figure 1.6.
1.6.2.1 Catalytic Triad Hydrogen-bonding Network and Proton Transfer
There has been much discussion about the mechanism used to effect hydrolysis in these enzymes. Originally, the double proton transfer or charge relay system was proposed, whereby a proton is abstracted from the histidine by the acid, which allows the deprotonated histidine to remove a proton from the catalytic serine, thus activating it (Blow, Birktoft et al. 1969). Although this mechanism keeps both the histidine and the aspartic acid neutral during catalysis, in order for the histidine to lose its proton to the acid, its pKa has to be lower than that of the acid. Although the pKa of a chemical
group can be altered by its environment, histidine, with a pKa of 6.08, would have to
have its pKa reduced to below that of aspartic acid (3.8) for this mechanism to be able
to occur.
More recently, the single proton transfer mechanism has been proposed. In this system, the catalytic histidine is stabilised by the charge on the acid residue. This does not involve a proton transfer between the histidine and the acid, but rather suggests that the function of the acid is to stabilise the charge on the histidine that is generated when it abstracts a proton from the serine during nucleophilic activation. The
interaction also plays a structural role in maintaining the alignment of the catalytic triad residues in an optimal position for catalysis. Experimental support for this mechanism comes from the fact that the pKa of the histidine in the serine protease
chymotrypsin is about 7.5, which means that it is likely to be protonated in the acidic environment in which chymotrypsin operates (Robillard and Shulman 1974). A compromise between the double and single proton relay systems is the low energy barrier hydrogen bond hypothesis, which involves a short strong hydrogen bond being formed between the histidine and the acid. There is some evidence for this, as, in
some structures, the sidechains of the acid and the histidine are less than 2.6 Ǻ apart.
This is also supported by nuclear magnetic resonance (NMR) spectroscopy, which shows that such a bond is present (Frey, Whitt et al. 1994). Although the histidine does technically retain its proton in a low energy barrier hydrogen bond system, the proton nonetheless is virtually shared between the acid and histidine residues. For an excellent discussion on the history and evidence for all three mechanisms, as well as serine proteases in general, the reader is directed to the review of Hedstrom (2002).
1.6.2.2 Nucleophilic Attack
Once activated, the nucleophile can then attack the scissile bond of the substrate, to form a tetrahedral enzyme–substrate oxyanion transition state, which is stabilised by hydrogen bonding between the oxyanion and the hydrogen bonds donated by the groups that form the oxyanion hole. The unstable tetrahedral intermediate then rapidly collapses into an acyl-enzyme intermediate. The bond that is formed between the nucleophile and the substrate during the nucleophilic attack remains, whereas the substrate scissile bond is broken during the collapse back to a trigonal centre, as the first reaction product is a better leaving group. This first product is then able to diffuse away from the enzyme.
1.6.2.3 Reaction Directionality
A mechanism must operate during the nucleophilic attack to prevent the tetrahedral state from simply collapsing back via the nucleophile and histidine base, which would result in breakage of the enzyme–substrate bond and lead to a futile cycle. It has long been hypothesised that this directionality must be orchestrated by hydrogen bonding, with the subsequent proton transfer being able to occur only in the direction that is favourable for the forward progress of catalysis. This results in a cycle of proton
Exactly how this occurs remained unclear for many years, despite the acid–base– nucleophile mechanism being well accepted. Even though a hydrogen bonding network between the triad members was proposed, early crystallographic studies showed that the nucleophile and the histidine base were too far apart and in
orientations that were not conducive for hydrogen bonds to form (Matthews, Alden et
al. 1977; James, Sielecki et al. 1980; Cohen, Silverton et al. 1981). Eventually, however, as more structures were solved to higher resolution, structures of serine proteases that showed that there was a hydrogen bond between the base and the
nucleophile were obtained (Bode, Chen et al. 1983; Tsukada and Blow 1985). This
was supported by an NMR experiment that also indicated the presence of the nucleophile–histidine bond (Bachovchin 1986). All these studies served to
demonstrate that some mobility of the catalytic residues is possible in serine-protease- type enzymes. Recent reports of crystallographic structures at atomic resolution have shown that there indeed appears to be a subtle parting of the nucleophile and the histidine during catalysis that assists the reaction to achieve a productive cycle (Radisky, Lee et al. 2006).
Some movement is also seen in the catalytic histidine in αβ hydrolase structures, with
the histidine in the structure of tabun-inhibited mouse acetylcholinesterase undergoing a displacement (Ekstrom, Akfur et al. 2006), whereas, in dienelactone hydrolase, the histidine becomes disordered when the enzyme is bound to phenylmethylsulphonyl fluoride (PMSF), suggesting that it is more mobile (Robinson, Edwards et al. 2000). Analysis of the structures of a number of αβ hydrolases has shown that it is not
uncommon for the distance between the nucleophile hydroxyl and the histidine Nε1 to
be too great for a hydrogen bond to form, implying that some kind of movement must occur for the enzyme catalytic system to be activated, and to encourage the reaction to proceed in a forward direction. Such a movement has been identified in Bacillus subtilis lipase, in which it appears that the nucleophile sidechain rotates into hydrogen-bonding range of the catalytic histidine, allowing enzyme activation (Kawasaki, Kondo et al. 2002).
The mechanism for disrupting this hydrogen-bonding network between the catalytic residues in αβ hydrolases was identified in this project, where the catalytic serine
sidechain of the esterase AZ4 was found to rotate back out of hydrogen-bonding range of the catalytic histidine during the nucleophilic attack (Section 5.5). This new
rotamer is distal enough from the histidine to break the hydrogen-bonding system, and could be a mechanism to prevent a futile collapse of the oxyanion.
1.6.2.4 Acyl-enzyme Intermediate
Once the scissile bond has broken, the first enzyme product (in the case of esterases, this is an alcohol) is released from the enzyme as it is no longer physically bonded to the remainder of the substrate. The remainder of the substrate is bound to the enzyme in an acyl-enzyme intermediate. The formation of the acyl-enzyme intermediate helps to lower the energy required for the reaction, as, if it was necessary to precisely orient two substrates (in this case, the acylglyceride substrate and the water acceptor), there would be a higher entropy barrier because of the necessity of positioning both molecules. In its acylated state, the enzyme is required to precisely position only the acceptor for deacylation to proceed, as the donor is relatively rigidly positioned by being covalently bound to the enzyme (Spector 1973; Silverman 2000).
1.6.2.5 Deacylation of Enzyme
A water molecule enters the catalytic site of the enzyme, and gets close enough to the catalytic histidine to donate a proton to it in a process that is similar to the process that occurs in the activation of the serine residue in the first step of catalysis. The activated water then carries out a second nucleophilic attack, this time on the acyl-enzyme intermediate. A second tetrahedral intermediate is formed, again stabilised by the oxyanion hole and the dipole from the N-terminus of helix C. This time, however, the tetrahedral intermediate collapses, breaking the bond between the substrate and the catalytic serine. The serine is then reprotonated by the proton that was donated to the histidine to complete the cycle. The carboxylic acid product can then diffuse away from the enzyme, allowing the catalytic cycle to begin again.
Asp O O His N N H H O Ser R2 O R1 O Asp O O H His N N H R2 O R1 O O Ser + Asp O O H R2 O R1 O O Ser His N N H Asp O O H H O R1 O O Ser His N N H Asp O O H His N N H H O R1 O O Ser + Asp O O H O Ser H O R1 O His N N H
1.
3.
5.
2.
4.
6.
Figure 1.6 Mechanism of ester hydrolysis.
Substrate binds and carbonyl carbon is attacked by the enzyme nucleophile (1) to form a tetrahedral oxyanionic intermediate (2). The intermediate decomposes to form an acylated enzyme intermediate (3), and the first product (alcohol) is released. Water binds and performs nucleophilic attack on the acyl-enzyme (4), which forms a second tetrahedral anion (5). This decomposes to break the acyl- enzyme bond and liberate the second (carboxylate) product (6). Diagram adapted from Figure 15.23 (Voet and Voet 2004).